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Brazilian Journal of Chemical Engineering

versão impressa ISSN 0104-6632

Braz. J. Chem. Eng. v. 14 n. 1 São Paulo Mar. 1997 



F. Merçon, V.L. Erbes, G.L. Sant’Anna Jr and R. Nobrega
Programa de Engenharia Química - COPPE - UFRJ - Cx. Postal 68502 - CEP 21945-970 - Rio de Janeiro, RJ - Brazil
Fax(55 21) 290-6626 Phone (021) 590-2297 E-mail:


(Received: August 12, 1996; Accepted: January 28, 1997)



Abstract - This work deals with enzymatic hydrolysis of babassu oil by immobilized lipase in membrane reactors of two types: a flat plate nylon membrane and a hollow fiber polyetherimide membrane on which surface commercial lipases were immobilized by adsorption. Experiments conducted in the hollow fiber reactor showed that during the immobilization step enzyme adsorption followed a sigmoid model, with a maximum adsorption equilibrium time of 30 minutes. Concerning the hydrodynamics of the liquid phases, the results indicate that main diffusional limitations occurred in the organic phase. The amount of protein immobilized and the maximum productivity were, respectively, 1.97 g/m2 and 44 m molH+/m2.s for the hollow fiber and 1.2 g/m2 and 56 m molH+/m2.s for the flat and plate membrane. Both reactors were able to perform the hydrolysis reaction, while maintaining absolute separation of the two phases by the membrane.
Keywords: Babassu oil, immobilized lipase, lipase, membrane reactor, oil hydrolysis.



The production of fatty acids through the hydrolysis of oils and fats is an important enzyme-catalyzed modification of lipids. The conventional methods of hydrolysis operate under high temperature (250° C) and pressure (50 atm) and these drastic conditions demand high energy consumption, as well as the formation of undesirable by-products. The enzymatic process is an alternative way to produce fatty acids, in which the enzyme-catalyzed hydrolysis is carried out under milder conditions, reducing the secondary reactions to a very low level due to the pronounced enzyme selectivity. Modifications of starch and proteins by enzymatic processes are well established on an industrial scale. However, the use of enzymes to modify lipids, despite the significant research effort made in this field, is still in a developing stage (Cuperus et al. 1993).

The hydrolysis of oils and fats is catalyzed by lipase (EC, which breaks the ester bonds of acylglycerides. Lipases have the particular characteristic of catalyzing the reaction only at the water/lipid interface. Thus, the surface area available for reaction is a crucial process efficiency factor. Both free enzymes in emulsion systems and immobilized enzymes were extensively studied to perform the hydrolysis of fats and oils (Linfield et al. 1984, Bühler and Wandrey 1987, Guit et al. 1991 and Cuperus et al. 1993).

In emulsion systems, the free enzyme spontaneously adsorbs onto the large area interface, which is generated by vigorous mixing. Although high reaction rates are achieved in these systems, some drawbacks may be pointed out: a fine emulsion should be maintained by intensive stirring, using emulsifiers and stabilizing agents; enzyme inactivation by high shear forces may be severe; and product separation and enzyme reuse is difficult to perform. In view of these drawbacks and considering that enzyme reuse is a key factor for process economical feasibility, the immobilized enzyme system, mainly the membrane reactor, appears to be a promising alternative.

Few works published in the literature deal with enzyme catalyzed hydrolysis of oils. The first reported work on the immobilized lipase membrane reactor was presented by Hoq et al. (1985), who used a flat plate membrane reactor. To improve the ratio membrane area/reactor volume, Pronk et al. (1988) used a hollow fiber membrane reactor for soybean oil hydrolysis. A comparative study between membrane and emulsion systems for the hydrolysis of triacetin by lipase from Candida cylindracea was carried out by Guit et al. (1991). These authors concluded that the specific enzyme activity in the membrane reactor was significantly higher than that observed in the emulsion reactor. Cuperus et al. (1993) studied the enzyme operational stability in a hollow fiber membrane reactor employing a hydrophilic membrane. Their system had a half-life of 160 days.

The aim of the present work was to study the hydrolysis of babassu oil by immobilized commercial lipases in two types of reactors, using flat plate and hollow fiber membrane modules. Enzyme adsorption and, mainly, the effect of liquid phase hydrodynamics on reaction rates were investigated.




Babassu oil (refined) was purchased from Dureino (Teresina-Piauí, Brazil). The microbial lipase (Mucor miehei), which is commercially available under the trade name of Lipozyme, was kindly provided by Novo Nordisk (Denmark). This is a 1,3 position specific lipase for the hydrolysis of triglycerides. The commercial lipase (Candida cylindracea), a position nonspecific enzyme, was kindly yielded by Sigma Co. (USA).


Experimental Apparatus

Two types of reactors were used i) a flat plate reactor and ii) a hollow fiber reactor. Two symmetrical rectangular plates (15 cm x 8 cm) and the membrane, forming two 1.2 mL volume compartments, constituted the flat plate reactor. A symmetrical hydrophobic nylon 66 membrane (Pall Ultrafine Ultrafiltration Company, USA) was used. The second membrane reactor was a PVC tube (20 cm length, 1.5 cm diameter) containing ten 0.3 cm internal radius hollow fibers (inner surface area of 36 cm2). These fibers were made of an asymmetrical, hydrophobic poly(etherimide) membrane, according to the procedure utilized by Oliveira et al. (1994).


Lipase Immobilization

For both reactors, the same immobilization procedure was used. It consisted of filtering a commercial lipase preparation dissolved in a buffer solution (citric acid 0.1 mol/L and sodium phosphate dibasic 0.2 mol/L, pH 7) through the membrane and thus allowing the adsorption of the enzyme on the membrane surface. The immobilization procedure was the same in both reactors. The commercial lipase preparation was dissolved in the buffer solution (citric acid 0.1 mol/L and sodium phosphate dibasic 0.2 mol/L, pH 7) and filtered through the membrane. Using this procedure, the enzyme spontaneously adsorbed on the membrane surface.


Reactional System

The experimental set-up is illustrated in Figure 1. The oil and the water phases were kept in different reservoirs and recirculated through the reactor by peristaltic pumps. In both cases the organic phase was in contact with the immobilized enzyme membrane side. The reactor and the reservoirs were kept at a constant temperature. The pressure difference between the liquid phases at the reactor inlet was adjusted and controlled to avoid their mixture.

Figure 1: Schematic diagram of the reactional system.


Analytical Methods


The amount of mmobilized protein was obtained from the difference between enzyme solution protein contents measured before and after the enzyme immobilization procedure. The protein determination was performed according to the classical method of Lowry et al. (1951). Due to its solubility, the fatty acids produced remain in the organic phase and are determined by tritation with a solution of NaOH 0.01 mol/L up to the end point of 9.6. As a 1,3 specific lipase was used, no glycerol production was expected in the present case. The lipolytic activity was obtained from the initial slope of the reaction plot, expressed in terms of International Unit (I.U.). One I.U. is defined as the amount of enzyme that liberates one m mol of acid per minute under standard conditions.




Adsorption of Mucor miehei Lipase over the Poly(etherimide) Hollow Fiber Membrane

The experimental results for different protein concentrations are presented in Figure 2. Data were obtained in the following conditions: 23 ° C, immobilization pH 7, volume of enzyme solution of 25 mL and flow rate of 3.2 mL.min-1. Results indicate that enzyme adsorption increases with the enzyme concentration reaching a limiting value, which corresponds to the adsorption equilibrium value for each case.

Considering that the equilibrium was practically reached just after 30 minutes, an adsorption isotherm was obtained as illustrated in Figure 3. The curve shape consists in a protein-polymer bond site saturation phenomenon, which, in this case, is described by a sigmoid model. The adsorption isotherm obtained is represented by Equation (1). Due to the absence of published data, comparison with other systems was not possible.



Ca = Adsorbed protein concentration (x 10-3 m g/cm2)
Cs = Protein concentration (solution) (m g/mL)

The module with immobilized enzyme was tested in the reactional system for the hydrolysis of babassu oil, under the following operational conditions: 45 ° C, oil volume of 8.8 mL, oil flow rate of 8.7 mL.min-1, buffer solution (pH 7) volume of 26.4 mL and buffer solution flow rate of 10.5 mL.min-1. The adsorption procedure was realized in a low temperature (23 ° C) to avoid the protein desorption during the immobilization process. However, the optimum temperature for the enzymatic reaction was 45 ° C (Merçon, 1994).. Even increasing the temperature it wasn’t detected any protein desorption during the reaction, due the bond protein-polymer.

The results, summarized in Table 1, indicate that in spite of the increase of both initial reaction rate and activity with the amount of immobilized protein a reduction of the specific activity was observed. Three assumptions could account for these results: i) several layers of protein could have been formed, hindering the access of the substrate to the inferior layers ii) the enzyme configuration was modified due to interactions caused by molecular proximity iii) immobilization of protein occurred far from the oil-water interface, restricting catalytic action to a small part of the total immobilized protein. This last assumption is based on the model proposed by Dalvie and Baltus (1992) for a different enzymatic system, as illustrated in Figure 4.


Hydrodynamics of the Flowing Phases in the Hollow Fiber Membrane Reactor

In order to study the effect of mass transfer on the initial hydrolysis rate, expressed as specific enzymatic activity, experiments were performed under different flow conditions, varying the Reynolds number of both liquid phases. This variation was achieved by operating with different liquid flow rates. To avoid the liquid phase mixture during the reaction, the aqueous flow rate was kept equal or higher than the organic phase flow rate. Consequently, the aqueous phase Reynolds number was equal or higher than the organic phase Reynolds Number.

Figure 5 shows that the specific activity significantly depends on the Reynolds number for the organic phase. The results indicate that an appreciable increase in activity is obtained by increasing the Reynolds number up to 10. However, an upper limit seems to exist, as increasing the Reynolds number beyond that value, only a negligible rise in activity was observed. The accumulation of fatty acids inside the membrane pores may cause enzyme inhibition. By improving mass transfer the fatty acids retrodiffusion is facilitated, rendering the microenvironment near the oil-water interface favorable for enzyme activity.

The results concerning the aqueous phase are presented in Table 2. It may be observed that in this case the flow conditions do not affect the enzymatic activity. Lipase inhibition by glycerol is a controversial matter as pointed out by Hoq et al. (1985). In the present work no glycerol production was expected because a 1,3 position specific lipase was used. As no accumulation of an inhibitory product occurred in the aqueous phase, the improvement of the mass transfer conditions did not affect the enzyme activity

Figure 2: Amount of immobilized protein as a function of the solution protein concentration (m g/mL) and time. Immobilization of Lipozyme on polyetherimide hollow fiber membrane.



Table 1: Experimental results for different concentrations of immobilized protein on the polyetherimide hollow fiber membrane reactor

Adsorved protein

Initial reaction rate


Specific activity
(mmolH+/min.g of protein adsorved)






Figure 3: Adsorption isotherm for Lipozyme on polyetherimide hollow fiber membrane. Equilibrium time was taken as 30 minutes (correlation coefficient = 0.996).


Figure 4: Schematic diagram of the enzymatic reaction in a cylindrical membrane pore.


Figure 5: Specific activity (mmol H+/min.g of protein) versus organic phase Reynolds number. Lipozyme immobilized on polyetherimide hollow fiber membrane.



Table 2: Experimental result for different aqueous phase Reynolds numbers on the polyetherimide hollow fiber membrane reactor

Reynolds number

Adsorved protein concentration

Specific activity
(mmolH+/min.g of protein adsorved)





Hydrodynamics of the Flowing Phases in the Flat Plate Nylon Membrane Reactor

To confirm the effect of mass transfer on the degree of hydrolysis, another reactor configuration (flat plate nylon membrane reactor) was used. The experiments were performed at different organic phase Reynolds numbers under the following experimental conditions: 45° C, oil volume of 51 mL, buffer solution (pH 6.4) volume of 90 mL. In a previous step, the amount of immobilized protein was 1.67 g/ m2 of membrane.

Figure 6 shows that the degree of hydrolysis rises with the organic phase Reynolds number. This seem to indicate that mass transfer in the organic phase was a limiting process step. Differences between the reaction progress curves were more significant for higher degree of hydrolysis, indicating that the removal of fatty acids at the neighborhood of the interface preserves the enzyme activity, thus increasing its stability. So, increasing the reactor hydrodynamics, it was possible to reach the maximum hydrolysis conversion (approximately 60%) for a 1,3 position specific lipase in a short time.

In order to achieve a complete hydrolysis, a nonspecific lipase, Candida cylindracea, was employed and the reactor was operated under the following conditions: 40 ° C, oil volume of 50 mL with a flow rate of 3.1 mL.min-1 (Reynolds number of 0.05), buffer solution (pH 7.0) volume of 100 mL and flow rate of 6.5 mL.min-1. The amount of protein immobilized was 1.2 g/m2 of membrane. The reaction progress curve in Figure 7 shows that it is possible to achieve the complete hydrolysis of the babassu oil in a membrane reactor. The excessive time necessary to achieve the complete hydrolysis is justified by the small relation membrane area / oil volume ratio and by the use of a closed circulation system. From the initial slope of this curve an initial lipolytic activity of 40.3 m mol of fatty acid.min-1 was calculated.

Figure 6: Degree of hydrolysis progress at different organic phase Reynolds numbers on the flat plate reactor with Lipozyme immobilized on a flat plate nylon membrane reactor.



Table 3: Performance results of membrane reactors for enzymatic hydrolysis of fats and oils







Amount of

m molH+

Hoq et al.





0.14 - 3.40


Taylor et al.







Pronk et al.




Cellulose acetate





Rhizopus sp










1.0 - 1.5









This work

Babassu oil

Mucor miehei



0.5 - 1.97


This work

Babassu oil


Flat plate





A survey of the published data on oil hydrolysis catalysed by lipase in membrane reactor is shown in Table 3. Even when operated with different enzymes and membranes, the productivities obtained in our reactors lie well within the range of the published data. An exception was the result obtained by Cuperus et al. (1993). These authors concluded that their higher productivity was consequence of the hydrophilicity of the membrane used in their system, which had an effect on the water activity near the active site of the immobilized enzyme.

Figure 7: Degree of hydrolysis progress of Candida cylindracea lipase immobilized on a nylon flat plate membrane reactor.



The lipase immobilized membrane reactor shown to be a promising process. Complete hydrolysis of babassu oil may be attained in immobilized lipase membrane reactors without mixing both liquid phases. Thus, downstream separation processes for fatty acids recovery may be either reduced or simplified.

Lipase immobilization on the hollow fiber membrane surface followed a sigmoid adsorption model. The adsorption equilibrium was reached in 15 to 20 minutes, resulting in an enzyme load in the range of 0.5 to 1.97 g of protein/m2.

The results support the motion that mass transfer in the organic phase was a limiting process step, while improvements in the aqueous phase hydrodynamics did not affect the specific enzyme activity. On the other hand, increasing the organic phase Reynolds number, there is an improvement in fatty acids removal from the interface neighborhood, thus increasing both lipase activity and stability.



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