Znd. Eng. Chem. Res. 1994,33,2591-2599
2691
Study and Comparison of Two Enzyme Membrane Reactors for Fatty Acids and Glycerol Production Raffaele Molinari,' Maria Elena Santoro, and Enrico Drioli Department of Chemical Engineering and Materials, University of Calabria, and Institute on Membranes and Chemical Reactors-CNR, 1-87030 Arcavacata di Rende (CS),Italy
Two enzyme membrane reactors (EMR), (i) with one substrate (olive oil) in a n oil-in-water emulsion (E-EMR) and (ii) with two separated liquid phases (oil and water) (TSLP-EMR), have been studied for the conversion of the triglycerides to fatty acids and glycerol. The enzyme was Candida cylindrucea lipase confined on the pressurized face or entrapped in the sponge side of capillary ultrafiltration membranes. Two methods for immobilizing the enzyme in the TSLPEMR were used: ultrafiltration on a virgin membrane and ultrafiltration on glutaraldehyde pretreated membranes. A multiple use of the reactor was obtained immobilizing the enzyme on the membrane preactivated with glutaraldehyde. The TSLP-EMR showed a specific activity of 0.529 mmol/(mgh) versus a specific activity of 0.170mmol/(mgh) of the E-EMR. The rate of fatty acid production in the TSLP-EMR was linear with time showing no enzyme deactivation in a n operating time of 80 h. The kinetics observed in the two reactors was different: a n equilibrium reaction product-inhibited for the E-EMR and a n apparent irreversible reaction of zero order for the TSLP-EMR. Taking into account that in the TSLP-EMR, compared to the E-EMR, (i)the specific activity was higher, (ii)the specific rate was constant with the time, and (iii) the two products were already separated after the reaction, the TSLP-EMR configuration seems the more convenient.
Introduction Recently technology of fats has been studied to analyze the possibility of replacing traditional chemical processes with biotechnological ones (Werdelman, 1982). Biological techniques, in fact, operate in mild conditions with low energy consumption and low pollution production. Renewable sources, such as vegetable oils, and fat surpluses can be generally used to transform their triglycerides into products of interest in various industrial sectors. First industrial fat splitting processes used biocatalysts, such as lipase from castor bean (Green, 1890)and from pancreas (Balls and Matlack, 19381,but no commercial application was made because of the sluggishness of the hydrolysis, its incompleteness, and general difficulty in engineering operations. Processes using or not using inorganic catalysts found more success on an industrial scale (e.g., Twitchell splitting in 1898; Colgate-Emery process in 1947) operating a t continuous or discontinuous mode, at temperatures in the range of 150-260 "Cand at 1.25.0 MPa pressures, reaching a degree of hydrolysis greater than 97-98% in periods of time from 3-4 to 24 h. The energy required in splitting processes is the highest in the oleochemistry industry. For example, in the Colgate-Emery process it is about 790 MJkg of splitted fat (Sonntag, 1989). As a result of the increasing costs for heat and electrical energy, after 1970,mild condition enzymatic methods for industrial fat splitting assumed importance on a world scale. In recent studies (Lieberman and Ollis, 1975;Kilara et al., 1977;Yamane et al., 1982;Han and Rhee, 19861, the use of lipase immobilized by entrapment or by adsorption idon solid porous particulates and in reversed micelles has been described. The catalyst particles can be used in fluidized beds, in stirred batch
* To whom correspondence should be addressed. 0888-5885/94/2633-2591$04.50/0
processes, in CSTRs, or in packed column reactors. The separation of products from the reaction mixture represents an additional cost for the process. Enzyme membrane reactors (EMR's) offer advantages with respect to conventional enzyme reactors (Drioli et al., 1975, 1976, 1990) for the membrane ability to operate simultaneously as catalyst support and as selective barrier. Various membrane materials (from hydrophobics to hydrophilics) and various configurations of EMR's, with the enzyme immobilized on the internal or the external membrane surface, have been reported in the literature (Hoq et al., 1985;Molinari and Drioli, 1986; Pronk et al., 1988) to accomplish the splitting process. In this paper two configurations of EMRs have been studied and compared to each other. In the first one (E-EMR), an emulsion (E) of the substrate is continuously recirculated inside the lumen of capillary ultrafiltration (UF) membranes. The enzyme is immobilized on the lumen-side surface. In the second configuration two (T) separated (S) liquid (L) phases (P) (TSLP-EMR) are continuously recirculated on the shell and lumen side of the membrane surfaces. The enzyme is immobilized on the sponge-side surface of capillary UF membranes. In both configurations at least one product leaves the reaction mixture through the membrane promoting the conversion from the thermodynamic viewpoint. A higher degree of conversion could be obtained with respect to other suggested bioreactors (Lieberman and Ollis, 1975; Kilara et al., 1977;Yamane et al., 1982;Han and Rhee, 1986). In this paper some theoretical aspects characterizing the two EMRs are described. An estimate to establish the form of the enzyme (above its solubility limit or below) in the E-EMR and some experimental results are reported. With regard to the TSLP-EMR the techniques for enzyme immobilization and the effects of the fluid dynamic parameters, like pressure and flow rate, are 0 1994 American Chemical Society
2592 Ind. Eng. Chem. Res., Vol. 33, No. 11, 1994 DENSE THIN LAYER
MESOPORES
of the membrane.
Figure
described. The performance of the two reactors, in terms of specific activity, is also reported. Theoretical Aspects Degree of Conversion and Performance of the Two Reactors. The hydrolytic reaction of the triglycerides is the following:
T
+ 3W t G + 3P
(1)
where T = triglycerides, W = water, G = glycerol, and P = fatty acids. As the volume during reaction is approximately constant with time, constant density for both the E-EMR and the TSLP-EMR can be assumed. Taking into account that triglyceride moles which have reacted at any time are stoichiometrically related to the moles of fatty acids produced by equation CTO- C , = (Cp - Cpo)/3 the degree of conversion of the key reactant (triglycerides), XT, can be expressed in terms of fatty acid concentration:
XT = (cp- cpO)/3cT(J
(2)
To compare the performance of the two reactors, from the various parameters (such as degree of conversion, volumetric rate of reaction, or productivity), specific rate (amount of product per unit time and per unit amount of enzyme) has been used since the physical characteristics of the reactors are different. In optimization and reaction model studies the effects of the main physical and chemical variables that influence specific rate can be linked in an “interaction logic diagram” (Fullbrook, 1983). Microenvironment and Mass Transfer in the Two Reactors. Triglyceride hydrolysis is a heterogeneous reaction that proceeds consecutively through diglycerides and monoglycerides formation with each step being reversible (Yamane et al., 1986). Numerous studies have demonstrated that in enzymatic lipolysis the reaction zone is a two-dimensional microenvironment,at the liquid-water interface, where lipase adsorbs and substrate and products partition from the bulk solution. When the enzyme is immobilized on membranes, the microenvironment is placed at the membrane-solution interface; therefore
the size and geometrical form of the reaction surface is known (it is not discontinuous as in a droplet system). The microenvironment is formed by cylindrical finite surfaces placed a t the lumen side, in the E-EMR, and at the sponge side in the TSLP-EMR. Its role is deeply different for the two configurations. In the E-EMR diffusive and convective flow in the radial direction cause mass transfer of the reagents to the catalytic surface. Radial convective flow is generated by an applied transmembrane pressure. The dominant flow depends on the value of the permeate flow rate. Water has two roles in the microenvironment: it is the reagent in excess and it is the component permeable through the membrane (hydrophilic). The concentration of water can thus be considered constant in the microenvironment, and water accounts for the transport of the glycerol produced. With regard to triglycerides, they are present in the E-EMR as small droplets, the size of which influences the diffusive transport to the microenvironment. A diffusive control by the substrate is expected considering also that the molecular size of the triglycerides is at least 3 times greater than the produced fatty acids and that the activation energy of enzymatic reactions is very low, generally lower than 75 kJ/mol (Malcata et al., 1992). In the TSLP-EMR the two reactants come in contact only at the membrane interface where simultaneously the enzymatic reaction and product separation occur. A net transmembrane pressure is not applied; therefore only diffusive mass transfer in the radial direction must be considered. In this case the two reactants (Figure 1) reach the microenvironment from two separate sides: triglycerides (as single molecules and not aggregates as in the E-EMR) must diffuse through the boundary layer; water, through the membrane pores. The diffusion time is L21D where L is the characteristic distance for the radial diffusion; this is the thickness of the boundary layer oil side, equal to about 0.5 mm. The membrane thickness is equal to 0.5 mm. Since Dwater Dtrigiycerides, the reaction rate controlling step is still expected to be triglyceride diffusion. Kinetics and Thermodynamics. The information reported in the literature (Hsu and Tsao, 1979; Pilone and Hanozet, 1983; Yamane et al., 1986; Ishikawa et al., 1987; Malcata et al., 1992),the experimental conditions used, and their results, allows some observations on the hydrolysis reaction and its kinetics to be made.
Ind. Eng. Chem. Res., Vol. 33, No. 11, 1994 2593 Three time ranges characterize any enzymatic reaction: reaction initiation, initial reaction, and main reaction. In the E-EMR the low solubility of the oil in water is enough to initiate triglyceride hydrolysis. In the second and third ranges, the produced fatty acids act as emulsification agents, so a high dispersion of the organic phase is obtained. Glycerol is removed from the reactions sites by diffusion or extraction into excess water; this last method is also used in the nonenzymatic reaction. However, also operating the reactor with glycerol continuously leaving the reaction mixture, because the glycerol permeates through the UF membrane, part of it is still present in the reaction system and it influences the degree of hydrolysis. The degree of hydrolysis will be practically independent of the type of fatty acid (Mueller and Holt, 1948) according t o the following formula (Bernardini, 1985): H = 100 - 8.OG, where H = splitting degree (weight percent) and G = glycerol concentration in the aqueous phase (weight percent); furthermore, it will not be affected by the temperature and pressure (Bernardini (1983, p 435; Sonntag (19891, p 33); thus Kes will be independent of these parameters. With regard to water, in microaqueous continuous environments (E-EMR and TSLP-EMR) it forms a monolayer near the active site of the enzyme, while in microaqueous discontinuous systems (droplets) it forms a pseudo-monolayer (which consists of clusters of water molecules around charged groups of enzyme) (Klibanov, 1989). The concentration of water may be taken as approximately constant in both cases if a reservoir of water molecules is available within a small distance of the active site. This situation is verified in the two reactors of this study. The above consideration permit eq 1 to be simplified with the following:
T;=rG+3P
(3)
Equation 3 is not described by a rate equation of the Michaelis-Menten type (Ishikawa et al., 1987) since it is an equilibrium. The form of the rate equation is more complex than that for a one-substrate one-product irreversible reaction. Uni-Quad mechanism, according to the King-Altman method (King and Altman, 1956), or mechanisms such as ping-pong, ordered, and random Bi-Bi or Uni-Bi type, are proposed in the literature (Malcata et al., 1992). The kinetic mechanism is also complicated by lipase inhibition caused by the produced fatty acids (Malcata et al., 1992): an inhibition mechanism has been recently proposed (Prazeres et al., 1992). Other factors influencing kinetics are the interfacial substrate concentration and the presence of organic diluents in the reacting system (Tsai and Chang, 1993). Taking into account that concentration of products and reverse reaction can be neglected in the initial step of the reaction, the initial rate has been used in this study. With these hypotheses the mechanism proposed by Michaelis-Menten (M-M) can be applicable (Hsu and Tsao, 1979) to eq 3:
T
+ E ATE -E + P k2
k-1
(4)
so that the initial reaction rate is related to the initial substrate concentration by the M-M equation:
where KMis the M-M constant (= (k-1
+ k2)/k1) and
Vm ( = k S o ) is the maximum reaction rate. Vm is also the maximum reaction rate of the forward reaction when the entire time course of the reaction (eq 3) is considered (Ishikawa et al., 1987). The above considerations permit easy quantification of the parameters KM and V, of the rate equation on the basis of the initial step of reaction. Values of Vm = 0.5 mmoY(Lmin) and KM = 0.85% v/v for the enzyme free in solution were estimated (Molinari and Drioli, 1987) by using eq 5. The integration of the complete form of the kinetic equation allows knowledge of the enzyme concentration and reaction time required to effect a desired conversion yield in the design of enzyme reactors (Fullbrook, 1983). In the TSLP-EMR both reactants are separated by the membrane. They are a t high concentration being practically in a pure form and have a large volume with respect to the reaction microenvironment. Assuming the diffusional external mass-transfer resistance of the two reactants, in the respective boundary layer, constant with time, their concentration can be assumed constant in the microenvironment because a large amount of water and triglyceride molecules are available in the bulk. This also means that all the enzyme is always saturated during the entire time course of the reaction. With these hypotheses only the second part of the reaction mechanism of eq 4 can be assumed, therefore,
By integrating eq 6 , it is easily obtained cp
- CpO = 3c,0.&= v,t
(7)
Plotting Cp or XTvs t, a straight line will fit the data if the hypotheses are consistent.
Materials and Methods The enzyme was lipase (E.C. 3.1.1.3) from Candida cylindracea (Sigma) with the following characteristics: 5 g of proteinl25 g of solids, 2975 unitdmg of proteins. The substrate was olive oil classified commercially as “extra virgin” (acidity 1%).All other chemicals were of analytical degree. Membranes used in the bioreactor were capillary tubes of aromatic polyamide (Forschungsinstitut Berghof, Germany) with an innedouter diameter of 1.5/2.5 mm. The membrane modules used for the E-EMR were made with three membranes of NMWCO of 10 kDa of 180 mm length, assembled in a Pyrex glass tube. A total inner membrane surface of 25.4 cm2was available. Modules used in the TSLP-EMR were of the same size as the previous, but with four membranes of 50 kDa NMWCO with a total outer membrane surface of 59.7 cm2. Concentration of fatty acids produced was measured by a kit for the determination of the nonesterified fatty acids in blood. It is based on the formation of a yellow complex detected by means of a spectrophotometer a t 480 nm. Glycerol concentration was measured using an enzymatic kit purchased from Boehringer Mannheim. The NADH produced in the last step is determined at 340 nm; it is stoichiometric with the glycerol. Protein concentration, depending on its value, was determined by absorbance a t 280 nm or by the Lowry test.
2594 Ind. Eng. Chem. Res., Vol. 33, No. 11, 1994
Except for some cases, indicated in the paper, the temperature in all the experiments was kept constant at 37 "C. The degree of conversion was calculated from eq 2, where the measured average value of Cpg was 0.75 mmoVL and the initial value of the triglyceride concentration CTO(mom) was calculated from the equation
V,dP
C,, = -
(8)
vbm
where V, = volume (mL) of the substrate utilized; d = density of the olive oil = 0.915 g/mL; p = average value of weight fraction of triglycerides = 0.984 g/g of olive oil; MW = average molecular weight of triglycerides in the olive oil = 872.3 &mol; v b = volume (L) of the solution, equal to the volume of the reacting mixture, for the E-EMR, and to the volume of the oil phase, for the TSLP-EMR. The activity of the enzyme was measured in batch tests in standard conditions. Typical conditions were 42 mL of Tris-HC1 buffer solution pH 6, 3 mL of olive oil (6% v/v), 3.7 mg of proteins (0.0074% w/v), and 0.05 g of arabic gum. First the arabic gum was added to the buffer in the stirred batch, then the oil, and after 2-3 min the enzyme previously dissolved in 5 mL of the same buffer. Just after enzyme addition the zero time was fmed, and a t stated times, samples were collected for 10-15 min and fatty acid concentrations were determined. Enzyme activity was determined as the slope of the linear part of the concentration-time curve. In order to establish the form of the enzyme a t steady state (soluble, completely confined, or partially confined, depending on enzyme amount and fluid dynamics), in the experimental conditions adopted in the E-EMR, the results of the analysis performed on an unstirred UF cell (Greco et al., 1979),by using a flat sheet membrane, have been used. The maximum enzyme concentration at the membrane surface is CEI
= N( v/ADE)
(9)
If, on the basis of the total amount of charged enzyme,
N , eq 9 predicts an unrealistic maximum concentration CEIexceeding its solubility (gelation concentration) CEG, this implies that a fraction N' of the total enzyme amount will be confined while the other fraction, say
N",still remains in the soluble form. The amount of enzyme which is still in soluble form can be evaluated as
N" = ACE$E/V
(10)
and the amount of confined enzyme as N' = N - N". The procedure used to covalently bind the enzyme t o the membrane using glutaraldehyde was the following: the membrane was washed with distilled water and then the permeate flow rate was measured at standard conditions. A solution of 500 mL of 2.5% v/v gldtaraldehyde in carbonate buffer 0.1 M pH 10 was recirculated at the shell side for 3-4 h at 34 "C and an inlet pressure of 10 kPa. After washing of the shell side and ultrafiltering with distilled water from the lumen to the shell, to remove the nonreacted glutaraldehyde, the enzyme solution, in phosphate b d e r 0.1 M pH 5 (optimum pH for the enzyme activity), was recirculated in the shell side for about 15 h a t an inlet pressure of 10 kPa and a temperature of 5-10 "C to reduce enzyme deactivation. The module, after washing with the buffer, at the same
0
........
0.0010.002 0.0050.010.02 0.05 0.1 0.2
0.5
1
Cb (%I
Figure 2. Permeate flux v8 protein concentration in the bulk (cb) (TMP = 60 kPa, T = 37 "C, axial flow rate = 1.0 Urnin).
temperature, to remove the nonbounded enzyme, was tested for catalytic activity.
Experimental Results and Discussion From an analysis of the literature on the various types of lipases, lipase enzyme from Candida cylindracea appeared the more interesting for the hydrolysis reaction because it has no positional specificity and splits almost completely the triglycerides in glycerol (Yamane et al., 1986; Bjorkling et al., 1991). E-EMR. In the experimental apparatus the emulsion, contained in a batch, was continuously recirculated into the lumen of hollow fiber membranes. The permeate was collected from the shell of the module applying a hydrostatic pressure. A makeup of buffer was added to the reservoir in order to keep the volume constant. Other details were described in previous papers (Molinari and Drioli, 1987; Molinari et al., 1988). In the startup of the bioreactor the enzyme was present in soluble form in a Tris-HC10.1 M pH 6 buffer solution; the enzymatic solution was recirculated into the capillary membranes, a t the established flow rate and pressure, in order to reach steady-state conditions. The amount of the enzyme varied from 20 to 315 mg (0.004-0.063% w/v in a total volume of 500 mL) depending on the type of experiment. M e r about 15 min the substrate was added and the zero time of the reaction was fixed. In order to analyze the data of the E-EMR, the form of the enzyme (soluble, completely confined, or partially confined on the membrane surface) needed t o be estimated. This was done by using eqs 9 and 10. The value of CEG was determined from Figure 2, where the transmembrane flux versus the logarithm of the initial enzyme concentration in the solution is reported; by using the film theory model a value of about 1%w/v (-10 mg/mL) was obtained. The membrane molecular weight cutoff (10 kDa) retained completely the enzyme (molecular weight 57-60 kDa (Patkar and Halkier, 1992; Alberghina et al., 1992)); therefore the enzyme amount N , initially charged into the reactor, was still present at the end of the transient period. In Table 1 the calculation of CEI,N', and N" is reported by using an estimated value of the diffusion coefficient of the lipase free in solution based on the radius of gyration (Tynand Gusek, 1990): DE = 7 x cm2/s. It can be seen CEIexceeds CEGin all the experimental conditions. The calculation of N' shows that practically all the enzyme is confined on the membrane surface if a deadend mode filtration system is employed (percentage
Ind. Eng. Chem. Res., Vol. 33, No. 11,1994 2596 Table 1. Calculation of the Percentage of Conflned Enzymea
0.004 0.008 0.063
20 40 315
516 1032 8130
10 10 10
0.387 0.387 0.387
19.613 98.06 39.613 99.03 314.613 99.88
Volume of enzyme solution = 500 & membrane surface area A = 25.4 cm2; permeate flow rate Q - 0.7 a m i n ; u = QdA = 4.59 x cmls; D E = 7 x lo-' cm5/s. -
Table 2. Calculation of the Specific Activity of the LiDase in the E-EMRa
0.004 0.008 0.063 a
0.22 0.42 0.99
6.82 13.02 30.69
40 80 630
0.170 0.163 0.049
VO= C~(dxddt)o, where Cm = 31 mmol/L. I
4 0
100
80
60
35 x
40
20
[fir -0
10
20
30
40
M
time (h)
Figure 3. Degree of conversion vs time and of amount of enzyme initially in the reservoir. ([olive oil] = 3% v/v, TMP = 60 kPa, axial flow rate = 1.6 Umin).
immobilized, (N'/W x 100, greater than 98%). Taking into account the significant difference in the fluid dynamics between the model system (unstirred UF cell) and the real system where a shear effect on the membrane surface (that influences the thickness of the confined enzyme) exists, the results found could be regarded as the limiting situation for an axial flow rate equal to zero. In the tangential cross-flow system, supposing a N" value 10 times higher, the amount of the confined enzyme is higher than 80%. Therefore, it can be assumed that the enzyme is prevalently confined on the membrane surface in the analysis of the E-EMR behavior. This theoretical result was also confirmed by previous experiments (Molinari et al., 1988) where the degree of conversion was not so sensitive to pressure change, according to the concentration polarization model (Porter, 1986), showing a little variation of the dynamic enzymatic layer thickness. The degree of conversion was maximum while operating with an axial flow rate of 1.6 Umin and transmembrane pressure 60 kPa; consequently these conditions were chosen in the following experiments. With the increase of the enzyme amount from 0.004% w/v to 0.063% w/v (Figure 31, the degree of conversion a t steady state increased reaching a value of about 90% in a reaction time less than 3 h. The equilibrium conversion in Figure 3 should not vary with the catalyst concentration but only with the time for reaching that conversion. The observed behavior might be attributed to the lipase inhibition, by the fatty acids produced, which decreases the amount of available active enzyme until a complete stop of the reaction. However, the inhibition is reversible: the equilibrium conversion was further increased from 30% to 48% in a test by doubling the initial substrate concentration of the reaction mixture after reaching the first steady-state conversion. The increase of the slope at t = 0 of the curves in Figure 3 is explained by eq 5: since CTOis the same for all the
Figure 4. Diafdtration of glycerol at constant volume (TMP = 70 kPa, axial flow rate = 1.6 Umin).
curves, uo = k'Eo therefore uo increases with the enzyme concentration. The constant k' = udEo is the observed specific activity of the immobilized enzyme (it takes into account the intrinsic specific activity and the diffusional and microenvironmental characteristics). The values of k', for different amounts of lipase used in the experiments, are reported in Table 2; uo is calculated from the equation uo = CTo(mdt)o, obtained taking the time derivative of eq 2 and using the identity u = -dCT/dt = dCp/3dt. (Wdt)o is calculated from Figure 3. In Table 2 it can be seen that k' decreases with increasing lipase concentration. This result could be explained supposing that neighboring molecules of immobilized lipase are excluded from contact with the substrate droplets, so only parts of the enzyme molecules are effectively available for the catalytic action. In addition the number of triglyceride molecules per enzyme molecule decreases from about 45 000 to 2870, corresponding to 0.004% and 0.063% w/v lipase, decreasing the probability of a catalytic event. One of the difficulties observed while operating such a system was the removal of glycerol from the recirculation reservoir. The glycerol was produced stoichiometrically with the fatty acids reaching in the reservoir a concentration of 25 mmol/L in a run performed with 0.063% w/v lipase initially free in solution, 3% v/v olive oil, 70 kPa pressure, and 1.6 Umin axial flow rate. The glycerol concentration in the permeate increased with the time starting from zero and reaching the same value present in the reaction mixture after 1.5 h of operation. The average permeate flow rate was low (0.5 mumin), and part of the glycerol still remained in the reservoir. For complete separation of the glycerol from the reaction mixture a diafiltration treatment at constant volume was used, Figure 4, by adding distillate water and operating for about 40 h. TSLP-EMR. One of the solutions to solve the problems mentioned before is the use of a two separated liquid phase EMR (Figure 5 ) . The two immiscible phases were continuously recirculated contacting only in the membrane module. Simultaneous enzymatic
2596 Ind. Eng. Chem. Res., Vol. 33, No. 11, 1994 PRESSURE GAUGE
8s
REACTOR
E
Y
GLYCEROL
PERISTALTIC ~
~
~~
Figure 5. Scheme of the laboratory plant of the two liquid phase enzyme membrane reactor. Table 3. Results of Various Tests of Lipase Immobilization by Dead-end Ultrafiltration I I1 I11 initial enzyme solution ( m L j 120 110 116 collectedpermeate(mL) 76 74 82 hold-up tube vol (mL) 44 36 34 initial [E]@g/mL) 46.43 45.92 54.08 collected perm. [E]@g/mL) 33.16 25 31.63 final tube [El@g/mL) 32.14 37.24 40.3 immobilization time (min) 85 67 65 initial perm. flow (mL'min) 2.5 1.9 2.8 final perm. flow (mL/min) 0.7 0.6 0.75 immobilized enzyme (mg) 1.64 1.86 2.31 TMP (kPa) 10 10 10 ~~~~~
OO-
Iv
v
~
113 67 46 49.47 22.63 35.79 79 1.6 0.7 2.43 10
110 65 45 49.47 27.89 38.42 120 0.9 0.5 1.9 10
reaction and product separation at the interface occur. The glycerol, soluble in the aqueous phase, and the fatty acids, soluble in the oil phase, were accumulated in the respective phases during the reaction; this reduces the costs for product separation. Since the reaction is product-inhibited,the inhibition will be reduced working with a large amount of the organic phase (pure oil); furtheremore the continuous removal of the products from the reaction microenvironment will shift more to the right side of the equilibrium equation. A membrane with a molecular weight cutoff of 50 kDa was used to obtain significant diffusive flux of reactants and products through the interface. The enzyme was immobilized on the sponge-side surface of the membranes during ultrafiltration of the enzymatic solution, from the outside to the inside of the lumen, choosing cross-flow mode and dead-end mode as the fluid path in the shell of the module. In the cross-flow mode 150 mL of lipase solution, 50 pg/mL (7.5 mg) in 0.1 M sodium phosphate buffer pH 5, were recirculated in the shell side of the module in tests performed a t inlet pressures of 20 and 5 kPa while recycling the permeate from the lumen side in the recirculation reservoir. Collecting samples from the reservoir, a decrease of the absorbance in the first 20 min was observed and then an increase was observed. Since the permeate was recycled in the reservoir, no concentration increase was observed, so this was attributed to the denaturation of the recirculating enzyme and was also confirmed by enzymatic tests on the solution in the reservoir a t the end of the immobilization. This phenomenon of denaturation during the immobilization by recirculation, probably caused by a shear effect and interactions a t air-water interfaces, was observed also by other authors (Narendranathan and Dunnill, 1982; Lee and Choo, 1989; Meireles et al., 1991). To overcome this difficulty a dead-end filtration technique was used. In the dead-end mode, solutions containing the enzyme (110-120 mL, 45.92-54.08 pg/ mL) were ultrafiltrated a t 10 kPa pressure. Permeate flow rate decreased from 2.5-2.8 mumin t o 0.5-0.75 mumin at the end of the immobilization. Depending on the run, the amount of immobilized enzyme ranged
20
60
40
I
I
80
100
tirne(h)
Figure 6. Fatty acid and glycerol concentration vs time. VR = volumetric rate; 12" = specific activity. (TMP = 10 kPa, T = 37 "C,2.31 mg of immobilized enzyme, 200 mg of Ca2+).
from 1.64 to 2.43 mg per module. In Table 3 a summary of five immobilization runs is reported. Each module was used as a catalytic unit in the scheme of Figure 5. The buffer solution (200 mL), at a flow rate of 167 mumin in the lumen side of the fibers, and the oil solution (225 mL), in the shell side a t a flow rate of 1.6 mumin, were continuously recirculated. The two phases were fed in cocurrent mode in order to have the pressure profiles both decreasing in the axial way, and the transmembrane pressure (TMP) was approximately constant in the axial direction. This operating choice was of crucial importance in order to avoid passage of water in the oil phase. In fact, since the membrane is a hydrophilic material, water is pushed, by capillary pressure, in the oil side according to the Young-Duprb equation: P, = (2y/u) cos 8 where P , is the capillary pressure, y the membrane-water interfacial tension, a the membrane pore size, and 8 the contact angle. The system worked with oil side pressures greater than those a t the water side, and with TMP increasing from 5, 10, to 20 kPa; this last value avoided passage of water in the oil phase and was used in all the tests. In this condition TMP 2 P, and the flux of permeated water is zero. This link between TMP and water flux also limits the choice of the lumen- and shell-side flow rates, because both the volume rate of flow and the pressure drop, causing the flow, depend on each other (e.g., Hagen-Poiseuille law Q = nAPR4/ 8pL, taking into account that generally in capillary membranes the flow is laminar). This means, for example, that for a fured oil-side flow rate, the aqueous phase flow rate cannot be higher than a certain value. The first tests of activity of the lipase immobilized by dead-end mode in the TSLP-EMR were run by using Ca2+ (1mg/mL buffer solution) as an activator. The results in Figure 6 show a linear increase of fatty acid and glycerol concentration in the oil phase and the aqueous phase, respectively. In another similar run, with 2.43 mg of immobilized enzyme, but without Ca2+, the same linear trend, but a higher volumetric reaction rate (VR),was observed (4.3 versus 1.95 mmol/(L-h)in the first case). This was attributed to the calcium ions present. The role of the calcium is discussed by many authors: a cofactor like Ca2+is reported to be required (Schandl and Pittner, 1984; Taylor et al., 1986) in order to increase the activity; this last, in fact, was tripled by addition of calcium ions (Molinari et al., 1988). However, calcium ion might not be a real activator and it probably removes the long chains of insoluble calcium salts, so this lipase does not require a cofactor that must
Ind. Eng. Chem. Res., Vol. 33, No. 11,1994 2897 I S T STEP
-
+ HOC (CH,),
- CHO
$
- H,O
CH -(CH,),
-
- CHO
2iE
Y
N
5
.-5 200
-
N = CH -(CHd, CHO
CH -(CH,),
+ E N = CH -(CH,),
- CHO
N
c
/p= 3.08rnmol/l.h
tsi 100 I
c
u
- CHO - H20
a
E
2ND STEP
N
300
5
CH -(CH,),
- CH
N-E
"0 N
CH -(CH,),
- CH = N-E
Figure 7. Reaction steps in covalent binding of lipase on the polyamide membrane by glutaraldehyde bridges.
be combined with the enzyme to express the catalytic action (Sonntag, 1989, p 54; Han and Rhee, 1986). Furthermore, Ca2+acts as inhibitor if present at greater concentration than the optimum (Molinari et al., 1988). For this reason, and taking into account that insoluble calcium soaps of fatty acids can plug the membrane pores, reducing mass transfer of reactants and products and consequently reducing the observed activity, in the following experiments Ca2+addition was avoided. Analyzing the results of two consecutive runs on the same TSLP-EMR, charged with an initial enzyme amount of 1.9 mg and operating at TMP = 20 kPa, a lower slope in the second run was obtained when the concentration of fatty acids versus time was plotted (VR = 4.5 mmoV(L-h)in the first run and VR = 2.11 mmoY (Lh) in the second run). To find if the decay of the volumetric reaction rate could be attributed to the leakage of the enzyme from the membrane or t o its denaturation, samples of oil (3 mL) were collected at the end of each run and tested with the standard test to measure whether the oil contains active enzyme. Active enzyme, free in the oil solution, was found. To reduce leakage, during reactor operation, the enzyme was bonded to the membrane surface by using glutaraldehyde as a bifunctional reagent (Onyezili, 1987; Kennedy et al., 1990). The technique is devided in two steps, Figure 7. In the first one the aldehyde group, in each glutaraldehyde molecule, links in accordance with the classical Schiff base mode with the amine groups on the membrane surface, leaving the distal aldehyde group available for covalent enzyme coupling. A pH 9-10 was used in order to have prevalently -NHz groups (low presence of -NH3+ groups) with glutaraldehyde activation also at the maximum. In the second step the distal aldehyde group covalently binds t o the -NH2 group of the lipase (€-aminoof L-lysine and N-terminus amino group). This phase is also favored by high pH, but in order to avoid enzyme denaturation, the optimum pH for the enzyme activity was chosen. When this technique is used, as a result of steric hindrance of the substrate with the catalytic triad (for the Candida cylindracea lipase this is probably Ser209-His449-Glu342 (Schrag et al., 1991)),part of the bound enzyme might be catalytically inactive. Therefore the active portion of the immobilized enzyme must be estimated after the immobilization (Clap& et al., 1988). At the end of membrane pretreatment, the technique of enzyme immobilization by dead-end UF was tested.
20
40 time (h)
60
80
Figure 8. Fatty acid concentration vs time in consecutive runs with the enzyme linked in the sponge side of themembranes (-1.9 mg of enzyme, TMP = 20 kPa, T = 37 "C).
A complete passage of the enzyme in the permeate was observed ultrafiltrating 100 mL of solution, 45 pg/mL. This behavior, observed only with the pretreated membranes, could be caused by a modification of the chemical-physical interactions of the membrane with the enzyme and to the short residence time for enzyme cross-linking a t the membrane wall. Therefore, the enzyme was immobilized by using the cross-flow filtration technique with permeate recycle, but a greater initial amount of enzyme, with respect to the dead-end UF technique, was used to reduce the percentage of denaturated enzyme. In Figure 8 the results of five consecutive runs, carried out on an EMR with the enzyme chemically immobilized, are reported. The EMR was prepared by recirculating 200 mL of enzymatic solution, 1 mg/mL (200 mg of initial enzyme), after the treatment of the membrane with glutaraldehyde. An amount of -1.9 mg of active enzyme was estimated using the VR values of runs on EMRs containing a known amount of enzyme immobilized by dead-end UF. Despite that the enzyme was covalently bonded to the membrane, from Figure 8 a continuous decrease of the volumetric reaction rate in multiple use of the reactor can be seen. Tests of activity in the oil solution at the end of each run showed negligible values, so no leakage of the enzyme was assumed. Also the thermal stress of the enzyme, between working condition (37 "C) and storage condition of the EMR (4 "C), could not be the origin of the decrease of the reaction rate because activity tests on the free enzyme in the solution, simulating the same thermal stress of the EMR, showed no deactivation. One of the causes of the activity decay could probably be the enzyme inhibition from the product because part of product still remains in the reaction microenvironment and does not completely diffuse in the fresh oil during the storage of the reactor. The behavior of the immobilized enzyme, as a function of the temperature, is reported in Figure 9. The estimated amount of enzyme was 1.3 mg after the immobilization on the pretreated membrane with an initial amount of 10 mg of enzyme (200 mL, 50 ,ug/mL). In Figure 9 two tests a t 37 "C were carried out, at the beginning and a t the end of the thermal stress, in order to compare the activity decay. It can be seen that enzyme inactivation is prevalent with respect the temperature effect: only at T = 50 "C the volumetric
2598 Ind. Eng. Chem. Res., Vol. 33, No. 11,1994
Acknowledgment VR
3.08 mmol /I h 0
1.BSrnmolAh
0.82m m l A h A
l.WmmDl/Ih
o 0.65 mmol /I h
I
5
10
15
nrne (h)
20
25
Figure 9. Fatty acid concentration vs time in consecutive runs at various temperatures with the enzyme linked in the sponge side of the membranes (-1.3 mg of enzyme, TMP = 20 kPa, T = 37 “C).
reaction rate is a little higher compared to the second run a t T = 45 “C. The data reported in Figures 8 and 9 were obtained with increasing oil flow rate from 1.35 to 2.15 mUmin and aqueous flow rate from 167 to 500 mLJmin. No variation of the slope (volumetric reaction rate) during each run was observed. This was attributed to the low influence of the axial flow rate on the boundary layer thickness, and hence the diffisional mass transfer in the two boundary layers, oil-membrane and watermembrane, was not improved. All the graphs of product concentration with time obtained in the TSLP-EMR show a linear trend in contrast to that obtained on the E-EMR. This behavior seems a confirmation of eq 7 described under Theoretical Aspects; therefore, an apparent irreversible reaction of zero order can be assumed for the TSLP-EMR. Calculation of the specific activity (k”),from the first run of various experimental tests, gave an average value of 0.529 mmol/(mgh). Comparing this value with the specific activity obtained in the E-EMR (k’),a specific activity 3 times higher for the TSLP-EMR can be observed.
Conclusions The immobilization of the enzyme in the TSLP-EMR by cross-flow filtration of the enzymatic solution, on the membrane preactivated with glutaraldehyde, gave a lower release of the enzyme in the oil phase compared to the non preactivated membranes. The rate of production of fatty acids was linear with time in the TSLPEMR, showing no deactivation of the enzyme during a run. The kinetic behavior of the two reactors was different due to the different role of the reaction microenvironment an equilibrium reaction product inhibited for the E-EMR and an apparent irreversible reaction of zero order for the TSLP-EMR have been observed. The performance of the two reactors, compared as specific activity, was better for the TSLP-EMR: a value of 0.529 mmoI4mgh) was obtained versus a value of 0.170mmol/(mgh) for the E-EMR. Taking into account that in the TSLP-EMR compared to the E-EMR (i)the specific activity is higher, (ii) the specific rate is constant in time, and (iii) the two products are already separated after the reaction, it seems more convenient t o use the TSLP-EMR configuration. A deeper understanding of the kinetics, the mechanism of enzyme deactivation in the reuse of the reactor, the dependence of membrane thickness, and the mass transfer through the interfaces on the observed specific activity will be helpful in the study on the TSLP-EMR.
We would like to thank the National Research Council of Italy (CNR), which partially supported this work within the Oriented Project “Chimica Fine 11”.
Nomenclature a = membrane pore radius, m A = membrane surface area, cm2 CEI= enzyme concentrationon the membrane surface, mg/ mL CEG= enzyme solubility, or gelation concentration of the enzyme, mgJmL CTO= initial triglyceride concentration, moVL CT = triglyceride concentration at time t, mol& CPO= initial fatty acid concentration, mom Cp = fatty acid concentration at time t, mom d = density, gJmL D = diffusion coefficient of water or triglycerides, cm2Js DE = lipase diffusion coefficient, cm2/s k‘ = observed specific activity of the enzyme immobilized in the E-EMR, mmol/(mg-h) k” = observed specific activity of the enzyme immobilized in the TSLP-EMR, mmol/(mgh) L = characteristic distance of diffusion, cm MW = molecular weight, kDa N = total enzyme amount, mg N‘ = confined enzyme amount, mg N” = soluble enzyme amount, mg NMWCO = nominal molecular weight cutoff, kDa p = weight fraction P, = capillary pressure, Pa Q = volumetric flow rate, mumin t = time, s TMP = transmembrane pressure, kPa u = permeate flux, cm/s V = volume, L XT= degree of conversion of the triglycerides y = interfacial tension, NJm 8 = contact angle, deg p = viscosity, Ndm2 Literature Cited Alberghina, L.; Lotti, M.; Fusetti, F.; Grandoni, R.; Longhi, S.; Brocca, S. Characterization of the multigene family coding for Candida cylindracea lipases. Presented at the CEC-International Workshop on Lipases: Structure, Mechanism and Genetic Engineering, Capri, Italy, Oct 1-3, 1992. Balls, A. K.; Matlack, M. B. J. Biol. Chem. 1938,123, 679;in Sonntag, ref 8. Bernardini, E. Fats splitting, fatty acid distillation, glycerine recovery and refining. In Oilseeds, oils and fats, 2nd ed.; B. E. Oil-Publishing House: Roma, Italy, 1985;Vol. 11, Chapter XV. Bjorkling, F.; Godtfredsen, S. E.; Kirk, 0. The future impe-t industrial lipases. Tibtech 1991,9, 360-363. Clap&, P.; Mata-Alvarez, J.; Garcia-Anton, J. M.; Reig, F.; Valencia, G. Estimating enzyme activities to design an immobilized reaction system. Chem. Biochem. Eng. &. 1988,2 (3),147-149. Drioli, E.; Gianfreda, L.; Palescandolo, R.; Scardi, V. Activity of acid-phosphatase as a gel layer overlaying an UF cellulose acetate membrane. Biotechnol. Bioeng. 1975,17 (91,1365. Drioli, E.; Ragosta, G.; Scardi, V. Ultrafiltration processing with enzyme-gel composite membranes. J . Membr. Sci. 1976,1,237. Drioli, E.; Iorio, G.; Catapano, G. Enzyme membrane reactors and membrane fermentors. In Handbook of Industrial Membrane Technology; Porter, M. C., Ed.; Noyes Data Corp.: Park Ridge, NJ, 1990; pp 401-481. M b m k , P. D. Kinetics. In Industrial Enzymology-The application of enzymes in industry; Godfrey, T., Reichelt, J., Eds.; Stockton Press: New York, NY,1983;pp 8-110. Greco, G., Jr.; Alfani, F.; Iorio, G.; Cantarella, M.; Formisano, A.; Gianfreda, L.; Palescandolo, R.; Scardi, V. Theoretical and ‘01
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