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Bioresource Technology 101 (2010) 95429549

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Analysis of a reactive extraction process for biodiesel production using a lipase immobilized on magnetic nanostructures
K.J. Dussan a, C.A. Cardona a,*, O.H. Giraldo b, L.F. Gutirrez a, V.H. Prez c
a

Departamento de Ingeniera Qumica, Universidad Nacional de Colombia Sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia Universidad Nacional de Colombia sede Manizales, Departamento de Fsica y Qumica, Carrera 27 No. 64-60 Manizales, Colombia c Universidade Estadual do Norte Fluminense Darcy Ribeiro, Centro de Cincias e Tecnologias Agropecurias CCTA, Laboratrio de Tecnologia de Alimentos LTA, Av. Alberto Lamego, 2000 Parque Califrnia, Campos dos Goytacazes/RJ, Brazil
b

a r t i c l e

i n f o

a b s t r a c t
Magnetic nanoparticles were prepared by coprecipitating Fe2+ and Fe3+ ions in a sodium hydroxide solution and used as support for lipase. The lipase-coated particles were applied in a reactive extraction process that allowed separation of the products formed during transesterication. Kinetics data for triolein and ethanol consumption during biodiesel (ethyl oleate) synthesis together with a thermodynamic phase equilibrium model (liquidliquid) were used for simulation of batch and continuous processes. The analysis demonstrated the possibility of applying this biocatalytic system in the reactive zone using external magnetic elds. This approach implies new advantages in efcient location and use of lipases in column reactors for producing biodiesel. 2010 Elsevier Ltd. All rights reserved.

Article history: Received 15 January 2010 Received in revised form 16 June 2010 Accepted 10 July 2010 Available online 14 July 2010 Keywords: Magnetic nanostructure Lipase Biodiesel Reactive extraction process Liquidliquid equilibrium

1. Introduction Biodiesel is obtained from a variety of vegetable sources such as soybean (Kaieda et al., 2001), sunower (Antoln et al., 2002; Mittelbach, 1999), cottonseed (Kse et al., 2002), rapeseed (Linko et al., 1998), and palm (Crabbe et al., 2001) among others. Biodiesel production from the above mentioned feedstock through transesterication with an alcohol requires acid or basic catalyst and includes pretreatment, transformation, distillation, neutralization, washing, purication, and evaporation steps. Purication involves high capital investment and high-energy consumption, which leads to high production costs. Recently, hybrid or simultaneous processes that combine reaction and separation operations in one unit have received much attention due to investment and energy cost savings, and the possibility of overcoming thermodynamic limitations imposed by reversible reactions or kinetic restrictions in irreversible reactions when separation of products is carried out in situ (Pai et al., 2002; Pisarenko et al., 2001). Reactive extraction is a process that involves reaction and separation of liquid phases in the same unit (Pai et al., 2002). Phase separation can occur naturally in the reactive system or can be
* Corresponding author. Tel.: +57 6 8879300x50417; fax: +57 6 8879300x50452. E-mail address: ccardonaal@unal.edu.co (C.A. Cardona). 0960-8524/$ - see front matter 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2010.07.044

introduced deliberately by adding a solvent (Samant and Ng, 1998). The technology increases yield and selectivity in a system with multiple reactions, reduces recycle streams and formation of waste streams (Cardona et al., 2006; Gutirrez et al., 2006; Gutirrez et al., 2005; Montoya et al., 2007), and facilitates purication of products that are difcult to separate by conventional technologies (Rivera and Cardona, 2004). Biodiesel production by reactive extraction is an option for reducing production costs and improving product yields (Cardona and Snchez, 2007; Gutirrez et al., 2009; Snchez and Cardona, 2008). The possibility of using enzymes in organic solvents has opened several biotransformation routes (Carrea and Riva, 2000; Claps et al., 1995). Frequently, lipase has been used for transesterication reactions (Abigor et al., 2000; Bela-Bako et al., 2002; Dizge et al., 2009; Orrego Alzate et al., 2005; Orrego Alzate and Valencia Rios, 2008). The most common drawback of the enzyme-based processes is the high enzyme cost; however, immobilization of lipases has been used to obtain reusable enzyme derivatives (Balcao et al., 1996; Hsu et al., 2002; Huang et al., 2006). Immobilization on magnetic nanostructures allows locating or concentrating immobilized enzymes at desired sites in a reactor by application of a magnetic eld (Dussn, 2008; Hristov, 2007; Hristov, 1996; Hristov, 1998; Penchev and Hristov, 1990). The aim of this work was to synthesize and characterize a system based on lipase immobilization on a magnetic nanostructure

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and to simulate a reactive extraction process involving magnetic elds for locating the biocatalyst at specic zones in the reactor. 2. Methods 2.1. Chemicals Iron(III) chloride hexahydrate (FeCl3) and iron(II) chloride tetrahydrate (FeCl2) were purchased from Carlo Erba and J.T. Baker, respectively. Tetraethyl orthosilicate (Si(OC2H5)4) and [3-(2-aminoethylamino)propyl]trimethoxysilane ((CH3O)3Si(CH2)3NHCH2CH2NH2) were purchased from Fluka. Ethyl acetate (CH3COOC2H5) was purchased from Riedel de Han. Methanol (CH3OH) was purchased from Fisher Chemicals. Sodium uoride (NaF) was purchased from AnalaR and ethyl alcohol (CH3CH2OH) from Carlo Erba. Glutaraldehyde solution, 50% in H2O (OHC(CH2)3CHO) was purchased from SigmaAldrich. Candida rugosa lipase (EC 3.1.1.3, type VII, solid, with a nominal specic lipolytic activity of 1104 U mg1, containing $18.26% protein based on the biuret protein assay) was obtained from Sigma Chemical Co. (St. Louis, MO, United States). Phosphate buffer solution (pH 7.0, 0.1 M) was prepared with sodium phosphate monobasic (NaH2PO4) and sodium phosphate dibasic (Na2HPO4). All the above-mentioned chemicals were of 99% or higher purity. 2.2. Methods 2.2.1. Synthesis of magnetic nanostructure Magnetite was made as described by Zeng et al. (2006) and Dussn (2008). For the preparation of magnetic carriers, 500 mL of 0.2 M FeCl2 and 500 mL of 0.3 M FeCl3 were mixed, stirred vigorously for 1 h, and 200 mL of 4 M NaOH was added dropwise to the mixture, resulting in a black precipitate. The precipitate was ltrated with qualitative paper lter under vacuum and washed three times with ethyl acetate. The solids were removed by magnetic separation and dried in an oven at a temperature of 313 K for 10 h. 2.2.2. Preparation of coated magnetic nanostructure Two grams of Fe3O4 (magnetite) was suspended in 100 mL distilled water. A mixture of 5 mL [3-(2-aminoethylamino)propyl]trimethoxysilane (APTS), 15 mL of methanol and 5 mL of sodium uoride (NaF) (1% w/w) aqueous solution was stirred for 10 min. Then 20 mL of tetraethyl orthosilicate (TEOS) were dropped slowly into the ask and stirred vigorously at room temperature for 24 h. The precipitate was collected, washed three times with ethanol and water, and dried in an oven at a temperature of 313 K for 10 h. 2.2.3. Lipase immobilization Lipase was immobilized as described by Zeng et al. (2006) and Dussn (2008) by adding 100 mL of glutaraldehyde to 4.4 g of coated particles and stirring at room temperature. Then, 700 mg of C. rugosa lipase dissolved in 100 mL of phosphate buffer solution (pH 7.0, 0.1 M) was added and stirred for 30 min at room temperature. The mixture was subjected to centrifugation at 3000g for 20 min, and the particles were washed three times with phosphate buffer solution (pH 7.0, 0.1 M) and dried in an oven at 303 K for 10 h. The immobilized enzyme system was synthesized four times, obtaining particles with the same physical and chemical characteristics. 2.2.4. Characterization Fourier transform infrared spectroscopy (FT-IR, PerkinElmer, model Spectrum BX) was used for studying chemical bonds between native and coated magnetic particles. Micrographs were

obtained with a scanning electron microscopy with X-ray microanalysis (SEM, Jeol JSM-5910LV). Thermal stability and weight loss proles were determined using a Q500-TA instruments unit. All TGA measurements were accomplished in nitrogen atmosphere and heating the sample was heated from 30 to 800 C with a heating rate of 10 C/min. The typical sample quantity was 2030 mg. DSC Q100-TA Instruments unit was used for the differential scanning calorimetric analyses. All DSC measurements were done in nitrogen atmosphere and the sample was heated from 33 to 600 C with a heating rate of 10 C/min. The typical sample quantity was 11 mg. 2.2.5. Lipase activity measurement In order to determine the enzymatic activity, esterication of oleic acid with butyl alcohol was followed. Reaction required 1.57 mL of oleic acid, 48 mL of hexane and 31 mg of enzyme. This mixture was prepared at 40 C. After preparing the enzyme solution, 0.46 mL butyl alcohol was added. The, oleic acid and butyl alcohol concentrations in hexane were of 0.1 N. One-milliliter samples were removed and the reaction was stopped by heating to 60 C for 1 min and dilution with 4 mL of ethanol. The samples were titrated with alcoholic potassium (0.01 N) using phenolphthalein as indicator. The purpose of this titration was to measure the quantity of oleic acid esteried. The activity is expressed as the amount of oleic acid esteried per minute (Dussn, 2008). This procedure was made for both free and immobilized enzyme. 2.2.6. Magnetic properties The magnetic properties of the immobilized particles were conrmed by applying an external magnetic eld with neodymium magnet. The magnet was triple coated (NiCuNi) and had maximum energy product (BHmax) ratings from 40 to 42 megaGauss Oersteds-MGOe (%13,000 Gauss) and a 353 K maximum operating temperature (e-Magnets UK, 2009). 2.2.7. ModELL-R software ModELL-R, developed by Gutirrez and Cardona (2006) and included in the Delphi package version 7.0 (Borland Software Corporation, USA), was used for solving batch and continuous reactive extraction processes. This software has been tested in acetate production and alcohol fermentation processes (Cardona and Snchez, 2007; Gutirrez, 2008; Gutirrez et al., 2006; Gutirrez et al., 2005; Gutirrez et al., 2009; Montoya et al., 2007; Rivera and Cardona, 2004; Snchez et al., 2006). 2.2.8. Modeling of reactor-extractor The reactive phase equilibrium is a useful tool for determining the viability of a process involving reactive extraction. The reactive liquidliquid equilibrium is related to systems in which two or more liquid phases and chemical reactions exist simultaneously. In this type of systems, rafnate and extract compositions correspond to both chemical and liquidliquid phase equilibrium subspaces. With the reaction kinetics or chemical equilibrium data, the liquidliquid-reactive equilibrium (LLRE) can be solved for a continuous or batch reactor. The model equations correspond to the mass balance around a control volume that corresponds to a reactor of complete mixture (CSTR) in the analyzed case. This reactor acts as a phase separator and hereinafter is denominated reactor-extractor (RE). RE represents a LLRE stage in which reagents react until reaching some extent of reaction (conversion) with a pseudo-initial composition. The term pseudo-initial applies to any composition obtained during reaction and immediately separated by liquid liquid extraction. After the steady state is reached, phase separation occurs eliminating the thermodynamic limit of chemical-reaction equilibrium because concentration elds became redistributed. Last

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allows new reagents re-concentration in the reactive zones increasing the driving force for reaction (process intensication in general). These stages of LLRE can be extended to systems with multiple stages turning the RE to a multi-stage reactor-extractor (MSRE). In this case, the mixing value reects the compromise between the need for mixing the reaction components and the need to allow phase separation. In the practice, phase separation in a MSRE is driven by the difference of densities among rafnates and extracts along the stages. This type of compact unit has been designed previously, and was successfully tested with basic and acid chemical catalysts and is currently being patented in Colombia (Gutirrez et al., 2007). The modeling of the continuous MSRE was based on the same equations that describe the continuous reactor-extractor. 2.2.8.1. Kinetics. Since no kinetic data on the alcoholysis of palm oil with ethanol using lipase from C. rugosa are available, data on alcoholysis by lipase from Mucor miehei reported by Oliveira and Alves (2000) were used for modeling and simulation purposes. The methodology has been discussed in detail in Faccio (2004) for lipase-mediated tranesterication. The reactions used in the model are represented in a single step:

For glycerin production:

dG 0 k1 TA3 k2 GEO3 dt

The mass balance equations used for a continuous reactorextractor are as follow: For triolein consumption:

T0 T

k1 TA3 k2 GEO3

where s is the residence time. T0 is the triolein initial molar concentration. For alcohol consumption:

A0 A

3k1 TA3 3k2 GEO3

10

where A0 is the initial molar alcohol concentration. For ethyl oleate production:

EO0 EO

3k1 TA3 3k2 GEO3

11

T 3A $ 3EO G

Lipase

where EO0 is the initial molar ethyl oleate concentration. For glycerin production:

where [T], [A], [G] and [EO] are triolein, ethanol, glycerin and ethyl oleate molar concentration in kmol/m3, respectively. As proposed by Oliveira and Alves (2000), the model is based on the following assumptions: (a) the reaction is reversible, (b) there are no losses of enzyme activity during the course of the reaction, (c) formation of mono- and diglycerides are not taken into account, and (d) mass transfer limitations in the reaction system were ruled out. Therefore, the reaction and separation times are assumed to be similar. From the stoichiometry, the following relationship between substrates and products was established:

G0 G

k1 TA3 k2 GEO3

12

where G0 is the initial molar glycerin concentration. 2.2.8.3. Liquidliquid equilibrium equation. Each component concentration is calculated using the following relation for phase equilibrium.

xs k i xi i

13

dT 1 dA dG 1 dEO dt 3 dt dt 3 dt
In addition, the kinetic constants are:
0 k1

where xs is the composition of component i in the solvent phase, ki i is the distribution coefcient and xi is the composition of component i in the phase where reaction occurs. 2.2.9. Simulation procedure With the aim of developing a model for describing both enzymatic process and liquidliquid extraction processes, the enzymatic kinetics was coupled with an extraction model. The liquid liquid equilibrium was described through an algorithm based on mass balance equations developed for an isothermal ash with two immiscible liquid phases. Activities of components in each phase were calculated by UNIFAC model, since this model has demonstrated to be, in this case, the most appropriate for describing the equilibrium when two or more liquid phases are present (Batista et al., 1999; Montoya et al., 2007). This algorithm was integrated into the ModELL software (Gutirrez and Cardona, 2006), which was designed for analysis and design of reactive separation processes. This software couples two convergence algorithms (NewtonRaphson and False Position Method) in order to calculate the liquid fraction of each phase (see Fig. 1) (Gutirrez, 2008; Gutirrez et al., 2005; Snchez et al., 2006). The methodology allows nding a limit for the solvent to feed ow ratio (R) at which the simultaneous process can be accomplished with external or internal solvent. In the software ModELL-R following assumptions are considered: the reaction occurs in a single phase. It means that reaction only occurs where the two reagents exist in any step and are well mixed. For calculation purposes, only one liquid phase was considered when the kinetics of reaction is solved. In addition it was assumed that any compositions corresponding to the liquidliquid equilibrium subspace were immediately redistributed along liquidliquid nodes into extract and rafnate uxes, that the reactor-extractor was stirred perfectly,

k1 k4 E expk3 A
1 1

k1 0:534 m3 =kmol3 h k2 1:54872 10


3 6 3

m =kmol3 h m3 =kmol
4 1

k3 1:6554 103 E 1:21 106

k4 158:94 m =kmol h

kmol=m3

where [E] is the enzyme concentration. 2.2.8.2. Mass balance equations. The mass balance equations used for a batch reactor-extractor are as follow: For triolein consumption:

dT 0 k1 TA3 k2 GEO3 dt
where k1 and k2 are kinetic parameters. For alcohol consumption:
0

dA 0 3k1 TA3 3k2 GEO3 dt


For ethyl oleate production:

dEO 0 3k1 TA3 3k2 GEO3 dt

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Fig. 1. Liquidliquid equilibrium algorithm using the kinetics of the reaction (Gutirrez and Cardona, 2006).

and that the reactor volume was sufcient to reach reaction equilibrium. The last assumption is based on the following reasoning. Residence time depends on volume/volumetric ow rates and during short cut calculations (refers to a method or procedure, used here, that is strongly based on known thermodynamic theories and reduces dramatically the time for calculations) the feed volumetric ow is xed and then it is assumed that reaction volume is sufcient to reach the desired conversion or equilibrium conditions avoiding the problem of residence time denition. The reaction rate decreases as the reaction reaches the equilibrium and the only form to compensate this situation is to increase the reactor volume in the same proportion. These assumptions are discussed in detail in previous studies (Dussn, 2008; Dussn et al., 2007; Dussn et al., 2006; Gutirrez, 2008; Gutirrez et al., 2006; Montoya et al., 2007; Pisarenko et al., 2001; Snchez et al., 2006). Triolein transesterication with ethanol was used as the most representative system for vegetable oils. Triolein is allowed to react with an excess of ethanol to form ethyl oleate (biodiesel) (Gutirrez et al., 2009; Van Gerpen et al., 2004). The immiscibility appears naturally in this system, therefore a solvent is not required for extracting the ethyl oleate and ethanol itself is be considered as an autosolvent. The use of triolein instead of vegetable oil for simulation purposes is based on the fact that vegetable oils are usually a mixture of more than ve triglycerides. Therefore, the number of components involved in a reaction mixture for producing biodiesel from any oil is at least 12 and the modeling and simulation of any reactive separation process is possible mathematically but not graphically as discussed by Pisarenko et al. (2001). The interaction parameters for the modied UNIFAC equation, developed by Batista et al. (1999) were used for the analysis of the triolein system. They adjusted new parameters for UNIFAC model for these fatty systems by minimization of the maximum likelihood objective function. For the UNIFAC model, the following groups were selected CH2COO, CH@CH, COOH, and OH and only

the interaction parameters with these groups were readjusted. The validation of the obtained parameters was made experimentally by these authors.

3. Results and discussion 3.1. Immobilized lipase on magnetic nanostructure FT-IR spectra of coated magnetic particles and immobilized enzyme demonstrated that adsorption bands are presented at 1068 cm1 due to the stretching vibration of SiO bond, and at 792 cm1 due to the bending vibration of NH2 groups. The existence of bands corresponding to the functional groups of TEOS (SiO) and APTS (NH2) chemicals used in the preparation revealed the existence of the coating. The characteristic bands of protein (i.e., lipase) at 1550 and 1400 cm1 were present in the immobilized lipase on magnetic nanostructure, conrming its binding. In addition, the absorption bands at 3400 and 1630 cm1 refer to the vibration of remaining water in samples. Bands at 2920 and 570 cm1 show the stretching vibration of CH and FeO bonds, respectively. SEM micrograph of coated magnetic particles showed agglomerations because the non-coated magnetic particles were not dispersed in an appropriate substance. Then, they tend to be attracted to crowd together themselves, and to generate spaces between non-coated magnetic particles. Finally, these spaces were lled when coating was accomplished, which suggested that magnetic particles were covered. The analysis of immobilized enzymes on the magnetic particles showed that the immobilization procedure causes uniformity in the surface particles. Thermal analysis of the particles using TGA and DSC showed an abrupt increase in energy of activation ($10 to $17 kcal/mol). The kinetic order of the thermal desorption reaction changed from one

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to two because of the change in the heat of wetting of the surface (Rudge et al., 2000; Zhuravlev, 2000; Zhuravlev, 2006). According to the DSC proles for the coated magnetic particles and immobilized enzyme, the temperature for thermal desorption was close to 185 C conrming a change in the structure of the material related to the water adsorption capacity (mass loss $10%). The same values have been reported before for these materials by Zhuravlev (2000). In the temperature range of 30200 C, an endothermic event occurs as the result of solvent evaporation (ethanol/water used in biocatalyst preparation), and removal of bound and unbound moisture according to the TGA and DSC data obtained in this work. This is equivalent to a weight loss of about 4%. At higher temperatures, heating induces water molecules release from the associated supercial hydroxyl groups and formation of siloxane links (Si OSi) which results in a denser and stable silica. Additionally, the immobilized enzymes were completely denatured at over 800 C and weight losses increased to 35%. Activity measurements showed a reduction of 20% in the activity of the immobilized enzyme with respect to the free enzyme. This can occur because the immobilized enzyme has less active sites after immobilization. Additionally, the magnetic properties were conrmed with excellent separation from the reaction medium using neodymium magnet (e-Magnets UK, 2009). Last, conrm the possibility of using this immobilized enzyme in a reactive extraction process for biodiesel production localizing it in the appropriate zone to optimize the production of this biofuel.

3.2. Reactive extraction process The thermodynamic liquidliquid equilibria at 313 K of the three possible ternary mixtures in the quaternary system (triolein, ethanol, ethyl oleate and glycerin) are shown in Fig. 2. All equilibria start at an ethyl oleate/glycerol/alcohol ratio of 1:0:0, indicating a high solubility of ethyl oleate/alcohol and a low solubility of ethyl oleate/glycerol. These data demonstrate that ethyl oleate separation from glycerin is feasible and that the two products will be separated immediately given their total immiscibility. The data also show that transesterication proceeds easier when the weight fraction of ethanol is under 0.67 and total miscibility is found. These results are consistent with data previously reported on the alcoholysis of sunower oil (Jachmanin et al., 2010; Makareviciene et al., 2005). The existence of these equilibria makes it possible to implement a reactive extraction process taking advantage of system characteristics for extracting the main product (ethyl oleate) without adding a solvent. The liquidliquid-reactive equilibrium was solved using the mass balance (Eqs. ()(5)(8)) and liquidliquid equilibrium equations (Eq. (13)) at a temperature of 313 K. The system behavior is shown in Fig. 3. High glycerin content mixture corresponds to rafnate and the high ethyl oleate content mixture to the extract. Rafnate is composed of glycerin and ethanol while the extract can be enriched with ethyl oleate. Once the system has been solved, it is possible to delimit the operation range for solvent to feed ow ratio (R).

Fig. 2. Liquidliquid equilibrium for the reaction system (weight fraction, temperature 313 K).

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Fig. 3. Liquidliquid reactive equilibrium for trioleinethanolglycerinethyl oleate system. Temperature 313 K. Fig. 5. Triolein conversion and ethyl oleate purity vs. molar ratio ethanol/triolein (R) for continuous process in the extract. Temperature 313 K, residence time 1 h and catalyst concentration 1.21 106 kmol/m3.

3.2.1. Batch process As the ethanol must be in excess, R is controlled by the feed ethanol concentration. The minimum value of R is given by the molar ratio ethanol/triolein in the transesterication reaction (R = 3). Fig. 4 shows the behavior of ethyl oleate purity in the light phase (extract) for different R values. It is observed that there exist a maximum in the molar concentration of ethyl oleate at R = 3.56. An excess of ethanol inhibits the reaction and the ethyl oleate concentration decreases. On the other hand, triolein conversion increases for higher R-values although tends to become constant at R = 5.0. In summary, the excess of ethanol favored the conversion of the transesterication reaction, but inhibited the enzyme, which involved a reduction in the ethyl oleate concentration. From the above mentioned situation can be concluded that the process of reactive extraction can be accomplished at relation R = 3.56 for avoiding the enzyme inhibition and obtaining a ethyl oleate molar concentration near to 95%.

and liquidliquid equilibrium (Eq. (13)). These equations were solved simultaneously with the aim of nding values of R where triolein conversion or ethyl oleate purity becomes maximum. Fig. 5 shows the triolein conversion and ethyl oleate purity prole when the molar ratio ethanol/triolein (R) is varied. A maximum of ethyl oleate purity was observed at R = 3.35 (Purity = 78.5%), but the triolein conversion was 84%. On the other hand, the higher triolein conversion (99.8%) was reached at R = 6.2. It is possible to say that the continuous reactive extraction can be carried out at R = 4.5 when the triolein conversion reached 95% and the ethyl oleate purity is 77%. 3.2.3. Multi-stage reactor-extractor (MSRE) The simulations showed in Fig. 6a demonstrated the possibility of producing an enriched ethyl oleate liquid phase (76.6% of ethyl oleate) and a glycerol liquid phase (45.6% of glycerol) in a multistage reactor-extractor, which was continuously fed with ethanol/triolein at a molar ratio (R) of 4.5. Experimental proles were obtained in previous studies (Gutirrez et al., 2007) for ve-stage MSRE using alkali instead of lipases and a palm oil mixture of triglycerides at the same R value. The results are shown in Fig. 6b and compared to the simulation results using lipases. These results agree with the simulation data showed in Fig. 6a. Even if alkali instead of enzyme were used, the discussed theoretical foundation of how MSRE can be applied for biodiesel production was experimentally conrmed. This is explained by the fact that lipases and alkali are catalysts performing the same task of accelerating the reaction depending on their concentration and location. Additionally, these results conrm the suitable usage of triolein instead of vegetable oils for modeling and simulation purposes. 3.3. Location of the biocatalyst using magnetic elds According to Fig. 6a, the maximum concentration of both reagents is achieved at stages 3 and 4. Therefore, catalyst should be located at these stages. Potentially, this can be easily achieved by applying a magnetic eld which would locate the immobilized enzyme at these stages. This technological approach is very innovative and has been used only for separation purposes (Nahmad Molinari et al., 2009; Oder, 1976; Oder and Jamison, 2010). Additionally, the intensities of the magnetic eld could be adjusted based on reagents concentrations at each stage. This

3.2.2. Continuous process The continuous reactor-extractor was solved using the mass balance (Eqs. (9)(12)), the kinetics of reaction (Eqs. (3) and (4))

Fig. 4. Triolein conversion and ethyl oleate purity vs. molar ratio ethanol/triolein (R) for batch process in the extract. Temperature 313 K, operation time 50 h and catalyst concentration 1.21 106 kmol/m3.

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Fig. 6. (a) Scheme of MSRE and (b) experimental concentration proles for alkali catalyzed production of ethyl oleate reported by Gutirrez et al. (2007).

approach would allow optimization of the biocatalyst mass at every stage avoid the possibility that excess catalyst might favor the reverse reaction when the concentration of products is higher than that of the reagents. The advantages of exact catalyst location have been discussed for reactive distillation processes by Pisarenko et al. (2001). Moreover, this process can be improved when the ethyl oleate liquid phase is continuously removed from the reactor-extractor and sent to a separation unit where ethanol is recovered and the glycerol phase is directed to another separation unit where another part of ethanol is recovered. This integrated conguration is shown in Fig. 7.

4. Conclusions The synthesis of a biocatalytic immobilized system with magnetic properties has been demonstrated experimentally. Additionally, the proposed methodology based on thermodynamics reduces calculation time for simulation of reactive extraction processes and allows a rapid understanding of process advantages and the best location of the biocatalytic system in the reactive extraction column. It is expected that reactive extraction can directly produce 77% ethyl oleate (biodiesel) using lower ethanol/triolein ratios compared to 3540% purity in a normal stirred reactor with a higher ethanol/triolein ratio. This work is a starting point for further developments in simulation and experimental use of magnetic elds for biocatalyst location into column reactors.

Acknowledgements The authors thank Dr. Medardo Prez, Advanced Microscopy Laboratory (National University of Colombia at Medellin) for technical assistance in SEM analyses, Dr. Andrs Rosales Rivera, Magnetism and Advanced Materials Laboratory (National University of Colombia at Manizales) for technical assistance in TGA, DTG and DSC analyses and Plasma Physics Laboratory (National University of Colombia at Manizales) for technical assistance in FT-IR analyses, Direccin de Investigaciones DIMA (National University of Colombia at Manizales) for nancial support.

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Fig. 7. Scheme of MSRE with ethanol recovery.

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