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Synthesis of Biosurfactants: Enzymatic Esterification of Diglycerol and Oleic Acid. 1. Kinetic Modeling
Mercedes Martnez, Rub en Oliveros, and Jos e Aracil*
Department of Chemical Engineering, Chemistry Faculty, Complutense University, 28040 Madrid, Spain ABSTRACT: The growing application of environmentally friendly products raised in the last few years has increased the interest in biodegradable nonionic surfactant products. As an example of these, polyglycerol fatty acid esters have a wide eld of application in many industries as additives of cosmetics, food, photography, inks, etc. Also, the huge range of possible raw materials for these products increases cited interest. The esterication of diglycerol with oleic acid using an enzyme (NOVOZYM-435) as catalyst was studied in this work. Several experiments were carried out at dierent temperatures, catalyst concentrations, and acid/alcohol molar ratios. A pseudo-second-order kinetic model, including Langmuir adsorption factors for the acid, was developed. The results show that this proposed kinetic model reproduces the experimental data with a maximum 10% error.

1. INTRODUCTION Polyglycerol fatty acid esters have a wide eld of application due to their behavior as nonionic surfactants, biodegradability, huge range of properties (liquids, solids, etc.), and the dierent raw materials available (vegetal, animal, saturated, unsaturated, etc.). The most important commercial products are the glycerol monostearate, monooleate, and monoricinoleate.1 The use of polyglycerol fatty acid esters implies many advantages over using glycerol fatty acid esters. In this way, the use of polyglycerol fatty acid esters in the cosmetic industry leads a huge revolution due to the high water adsorption of diglycerol, which helps to reduce the fatty sensation of glycerol. In the food industry, the use of these esters as additives in the E.U. is regulated in the European Directive 95/2/ EC. These esters also have application in the lubricant (due to their biodegradability), polymer, and ink industries. A growing interest in the biodiesel industry has arisen in the last few years because of the environmental implications of this non-fossil based fuel. Excess of raw materials, biodegradability, and social impact make this industrial area one of the most interesting in the near future.2 One of the critical factors for the viability of this process is the benet of the glycerol obtained as coproduct. Several works have been published on the esterication of glycerol and polyglycerol with fatty acids, looking for selective processes with high region-selectivity in which the catalyst can be easily recovered and, therefore, the energy consumption reduced. Akg ul researched the synthesis of glycerol with oleic acid using clinoptilolite as a heterogeneous catalyst.3 Another approach to the problem uses anion exchange resins to produce the condensation of glycidol and oleic acid.4 However, the most suitable exploitation for this coproduct is the enzymatic esterication with fatty acids or the polymerization and later enzymatic esterication with fatty acids in order to obtain glycerol and polyglycerol fatty acid esters.5 The high price and the wide application range make this alternative the most suitable one in the Mediterranean European countries, where high fatty acid productions are achieved thanks to the modied seeds of cereals such as soy and sunower, that can produce oil which has oleic acid content greater than 80%.6 An example of
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this fatty acid production is the new plantation of 30.000 ha of sunower seed genetically treated for the production of high oleic acid, located in Spain. In this work, the esterication of diglycerol with oleic acid is proposed. Glycerol, obtained as coproduct in the biodiesel production process, would be used for the production of the diglycerol. On the other hand, this alternative allows the development of a national raw material (high oleic acid), which emphasizes the economical interest in the process. The enzymatic esterication of glycerol and polyglycerol with fatty acids has been carried out using dierent lipases as catalysts in solvent free systems.7,8 When using oleic acid as reagent, the best results in acid conversion and monoester selectivity have been achieved with the commercial lipase Novozym-435 as catalyst.9 Another lipase, Lipozyme-IM, has been used as catalyst in these processes with dierent results. However, to the best of our knowledge, there is no information about the kinetics of the esterication reaction that would eventually become necessary for an industrial production. The objective of the present work is the study of the enzymatic esterication of diglycerol with oleic acid using Novozym-435 and Lipozyme and IM as catalysts. The development of a kinetic model10 and the process optimization, using the central composite design methodology,11,12 are also objectives in the second part of this research. Calculations were performed using experimental data obtained under dierent catalyst concentrations, acid/alcohol molar ratios, and temperatures.

2. EXPERIMENTAL SECTION
2.1. Raw Materials. Diglycerol of purity >98% (w/w) was supplied by Solvay Qumica S.L. (Spain), and oleic acid of
Received: December 22, 2010 Accepted: April 27, 2011 Revised: April 25, 2011 Published: April 27, 2011
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Industrial & Engineering Chemistry Research purity >98% (w/w) was supplied by Henkel Iberica. The catalysts used were immobilized thermostable lipases, Novozym,435 and Lipozyme-IM, which were particularly useful for the synthesis of esters. This catalytic system was kindly supplied by Novo Nordisk Bioindustry S.A. The main enzyme was a triacylglycerol hydrolase, E.C. 3.1.1.3, which simultaneously acts as an eective carboxylesterase. The positional specicity of this system depends on the reactants. In some reactions, it shows 1,3 positional specicity, whereas, in other reactions, the lipase functions as a nonspecic lipase. In the manufacture of Novozym 435, recombinant DNA technology was used. The gene coding for the lipase was transferred from a selected strain of Candida antarctica to the host organism Aspergillus oryzae. The enzyme obtained by this procedure was immobilized on a macroporous acrylic resin. The nal product consists of bead-shaped particles with a diameter of 0.3 mm. The bulk density of the catalytic system is approximately 430 kg/m3. The ester synthesis activity of Novozym 435 was 7000 PLU/g, where one propyl laurate unit (1 PLU) is dened as 1 mol of lauric acid converted to propyl laurate per minute per gram of enzyme under standard conditions.13 Lipozyme IM is a nonspecic triacylglycerol lipase from Mucor miehei. The water content of this enzyme is 12 wt %/wt. The activity of the enzyme preparation was 60 batch interesterication units (BIU). One batch interesterication unit is dened as 1 mol of palmitic acid incorporated into triolein per minute at standard conditions. The rest of the chemicals and solvents used for the chromatographic analysis and in the recovery process of the catalyst were supplied by Aldrich and Panreac, Spain. 2.2. Equipment. Experiments were carried out in a completely stirred tank reactor (STR) of 500 cm3 volume, 10 cm height, and 8 cm diameter. The reactor was equipped with stationary baffles attached along the circumference. A marine-type propeller was employed. The impeller speed was set at 0.035 Np to maintain the catalyst particles in good condition and because no significant influence of mass transfer on the process has been observed between 0.02 and 0.04 Np. A temperature recorder and controller and a speed controller were provided. The desired working pressure was maintained with a vacuum pump. This permitted ready elimination of water from the system in the range of temperature studied, without signicant variations of the viscosity of the liquid phase or reaction volume. The reactor was immersed into a thermostatic bath capable of maintaining the reaction temperature within (0.1 C of the set value by means of an electrical device connected to a PID controller. 2.3. Analytical Method. Reaction products were monitored by gas chromatography/mass spectrometry (GC/MS) and quantitatively determined by capillary column gas chromatography. The GC/MS data were obtained on a 6890S HewlettPackard instrument. Gas chromatography was performed on a fused-silica capillary column (OV-1, 12 m 0.31 mm i.d., 0.17 m film). A Hewlett-Packard gas chromatograph was equipped for slit-splitless injections (30 s). The GC/MS operating conditions were as follows: ionization energy, 70 eV; scan speed, 1100 amu 3 s; mass range, 40400 amu; data treated with a HewlettPackard 9825B computer. The GC column oven temperature was held at 170 C for 5 min and then raised at 10 C/min to 270 C, and maintained at that temperature until all components had eluted. Quantitative gas chromatography analyses were performed on a HewlettPackard 5890 Series II instrument using the column and

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Table 1. Physicochemical Characteristics of the Surfactant

physicochemical characteristics skin irritation (mN/m) (mN/m) cmc (mmol/L) slightly irritating 27 5.5 0.12

Table 2. Experimental Conditions

operating temperature (K) catalyst concentration (% wt) diglycerol:oleic acid molar ratio (wt:wt) stirring (Np) pressure (Pa) 338.15 343.15 348.15 0135 3:1 1:1 1:3 0.035 8.000

conditions described above. A Hewlett-Packard 3396A integrator was connected to the chromatograph. The detector was an FID type at 270 C, and the injection system was splitless. The carrier gas was helium, and the ow rate was 0.65 mL/min. Identication of the products of the reaction mixture was performed by thin layer chromatography (TLC) using silica gel plates supplied by Merck. Products were separated with chloroform/acetone (90:10 v/v) and visualized with sulphuric acid/ water (10:90 v/v). Only oleic acid (Rf = 0.7) and monoester (Rf = 0.31) were detected while no glycerol trioleate was found in the sample. Several physicochemical characteristics of the produced surfactant (dyglicerol monooleate) have been determined. Skin irritation potential was measured by in vitro HET-CAM (hens egg test on the chorionallantoic membrane). Surface tension with determination of the CMC has been carried out using the De No uy method. Interfacial tension was determined by the spinning drop method. Results are shown in Table 1. 2.4. Procedure. The reactants, oleic acid and diglycerol, were added to the reactor, and the stirring was started. When the desired temperature was reached, the catalyst was added, and the vacuum pump was turned on. The reactants were stirred during 4 h. Samples were taken at regular intervals and at the end of the reaction, and analyzed by gas chromatography. During the experiments, the following variables remained constant: acid/ alcohol molar ratio, temperature, pressure/ and agitation speed. Before an experiment was started, the system was flushed with nitrogen.

3. RESULTS AND DISCUSSION Thirteen experiments were carried out to determine the best catalyst and the inuence of the temperature, catalyst concentration, and diglycerol/oleic acid molar ratio on the reaction. Experimental conditions are shown in Table 2. 3.1. Previous Experiments. 3.1.1. Catalyst Selection. Figure 1 shows the typical curves of experimental conversion and selectivity versus time obtained for the different catalysts, setting the temperature at 348.15 K, the catalyst concentration at 3% of the total substrate weight, and the diglycerol/oleic acid molar ratio at 1:1. After this study, Novozym-435 was chosen as the best catalyst for this process because of the highest conversion (5%) and selectivity (8 times) obtained in comparison to the other catalyst tested (Lipozyme-IM).
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Figure 3. Study of internal diusion. Inuence of particle size. Catalyst conc = 3% of Novozym 435; T = 343,15 K; reactants molar ratio = 1.

Figure 1. Inuence of catalytic system (a) on acid conversion and (b) on monoester selectivity.

Figure 2. Study of external diusion. Inuence of stirring speed. Catalyst conc = 3% of Novozym 435; T = 343.15 K; reactants molar ratio = 1.

Figure 4. Inuence of temperature on (a) acid conversion and (b) on monoester selectivity.

3.1.2. Influence of Stirring Speed. Preliminary experiments were carried out at stirring speeds between 0.02 and 0.14 Np. Figure 2 shows the initial reaction rate vs power number (Np), setting the temperature at 343.15 K, the catalyst concentration at 3%, and the diglycerol/oleic acid molar ratio at 1:1. At lower values of power number (high stirring speeds), no influence on the initial reaction rate can be observed. This behavior indicates that at lower stirring speeds there is mass transfer dependence because of external diffusion mechanisms. However, no mass transfer limitation was detected at a power number below 0.04 Np. Therefore, the stirring speed was fixed at 0.035 Np. 3.1.3. Influence of Particle Size. Preliminary experiments were also carried out with a catalyst particle size range between 0.32 and 0.8 mm. Figure 3 shows the initial reaction rate vs particle size, using all the other variables at fixed values. The dependence of acid conversion on particle diameter can be observed. Differences between lower and upper sizes can produce almost an increase of 20% on acid conversion. In fact, mass transfer

limitations take place, probably due to internal diffusion phenomena. However, mass transfer limitations can be neglected for catalyst particle diameters below 0.55 mm. Therefore, all the experiments were carried out using those particles found in the sieve between 0.32 and 0.55 mm. 3.2. Kinetic Experiments. 3.2.1. Effect of Temperature. The effect of the temperature was studied by setting the catalyst concentration at 3% and the diglycerol/oleic acid molar ratio at 1:1. The results are shown in Figure 4. As is shown, an increase in the operating temperature implies an increase in the acid conversion and a decrease in the monoester selectivity. Maximum monoester selectivity was achieved at the less operating temperature. This eect is due to the lower mobility of the molecules and the higher energy required for the reaction at diglycerol tertiary carbons. The eect of the temperature was found to be similar for all catalyst concentrations and initial molar ratios.
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Figure 5. Inuence of diglycerol/acid molar ratio (a) on acid conversion and (b) on monoester selectivity.

Figure 6. Inuence of initial concentration of catalyst (a) on acid conversion and (b) on monoester selectivity.

3.2.2. Effect of the Initial Molar Ratio of Reactants. The effect of the reactants initial molar ratio was studied by setting the catalyst concentration at 3% and the temperature at 338.15 K. The results are shown in Figure 5. As expected, an increase in the diglycerol/oleic acid initial molar ratio implies an increase in the acid conversion and a decrease in the monoester selectivity due to the excess of diglycerol present in the reactor. The higher monoester selectivity was achieved when the molar ratio was set at 1:1. The acid conversion gap between the dierent catalyst concentrations is practically the same; this implies that the oleic acid adsorption process in the catalyst is not signicant at the operation conditions in this process. 3.2.3. Effect of the Initial Catalyst Concentration. The effect of the initial catalyst concentration was studied by setting the diglycerol/oleic acid initial molar ratio at 1:1 and the temperature at 348.15 K. The results are shown in Figure 6, where the evolution of both acid conversion and selectivity with reaction time can be observed as a function of initial catalyst concentration. The inuence, as expected, implies an increase in the reaction rate and monoester selectivity when the catalyst concentration increases. 3.2.4. Kinetic Model. Experimental results and previous works indicate that the kinetics of these esterification processes can be described by an irreversible second order power model.1,11,12 Also, the shape of the monoester selectivity curves (Figures 4b, 5b, and 6b), where a maximum was found, showed a series parallel mechanism. No significant amounts of diglycerol trioleate were detected. oleic acid diglycerol f monooleate water monooleate oleic acid f dioleate water 3.2.5. Effect of the Nonenzymatic Process. In order to estimate the influence of the nonenzymatic process, an experiment

Figure 7. Linear dependence of the kinetic model with catalyst concentration at dierent temperatures.

without catalyst was carried out at 348.15 K. The conclusion was that no nonenzymatic reaction took place, obtaining practically zero conversion to ester. This allowed us to take into consideration only the enzymatic process in the kinetic model. 3.2.6. Effect of the Catalyst Concentration on the Kinetic Model. To proceed with the kinetic study of the process, it is necessary to estimate the dependence of the reaction rate on the catalyst concentration. As is deduced from Figure 6a and shown in Figure 7, a linear relation between both parameters was detected. The evolution of the maximum reaction rate with the initial concentration of immobilized enzyme was almost linear for the three tested temperatures. This behavior made it possible to consider the catalyst concentration as a linear parameter in the kinetic model. 3.2.7. Irreversible Process Study. Assuming a second order reaction (one for each reactant), the linearity between the reaction rate and the catalyst concentration, an irreversible
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Industrial & Engineering Chemistry Research Table 3. Kinetic Constant Values: k (L 3 mol1 3 min1 3 gCAT1)
integral method T (K) 338.15 343.15 348.15 103k 4.6 5.7 8.3 103 1.8 2.8 4.8 dierential method 103k 6.5 7.9 13.1 103 3.2 5.2 8.2

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process, and a negligible extension of the reversible reaction, the kinetic model proposed is as follows: rAO 1 dCAO kCAO CDGL W dt 1

where CAO = CDGL due to the 1:1 initial molar ratio between both reactants. By using the integral method XAO kCAO 0 t 1 XAO 2

The kinetic parameters k and Ea were estimated using both the integral method and the initial reaction rate method (dierential method). The reaction rate kinetic constant was estimated by using a nonlinear regression method. The kinetic constant was calculated for each experiment, divided in series at constant temperature and in series at constant catalyst concentration. The values obtained are shown in Table 3. By applying the Arrhenius equation to the results obtained using the integral method, k0 and Ea were estimated to be Ea = 6.1 104 J 3 mol1 and k0 = 1.6 107 L 3 mol1 3 min1 3 gCAT1. According to the experimental data, the model proposed appears to be consistent with the following expression: 4 3 rAO 1:6 107 e6:110 =RT CAO CDGL Figure 8a shows the residual analysis of the process. As is shown in this gure, a trend to negative residual values at large time was detected. This eect could be due to an adsorption process of the diglycerol monoester in the catalyst. In order to improve the kinetic model taking into consideration this adsorption process, a dierent model was proposed. 3.2.8. Adsorption Kinetic Model. The proposed model was the following rAO kWC2 AO CME 1 KME Ccat 4

Figure 8. Residual analysis: (a) for the single second order kinetic model and (b) for the kinetic model including adsorption terms.

Table 4. Adsorption Constant Values: KME (gCAT 3 gME1)

T (K)\cat 338.15 343.15 348.15 1% 3.60 102 1.61 10 7.29 103
2

3% 6.68 102 1.80 10 2.40 103

2

5% 4.26 103 1.90 102 4.10 103

Applying Vant Hos equation to the experimental results obtained, KMeo and H were estimated to be KMEo = 9.8 109 gCAT 3 gME1 and H = 8.2 kJ 3 mol1. Applying Arrhenius equation to the results obtained using the initial reaction rate method, k0 and EA were estimated to be EA = 6.1 104 J 3 mol1 and k0 = 2.6 107 L 3 mol1 3 min1 3 gCAT1. According to the experimental data, the model proposed appears to be consistent with the following expression: 4 2:6 107 e6:110 =RT WC2 AO 6 rAO 3 1 9:8 109 e8:210 =RT CME =Ccat Figure 8b shows the residual analysis of the process. As is shown in this gure, no trend was detected. Also, the correlation coecients obtained show a good t for the proposed model, with an average error minor than 3%.

The adsorption constant was estimated using the kinetic parameters, k, estimated before by using the initial reaction rate and a nonlinear regression method with the following expression (deduced from eq 4):   kW CAO 0 1 XAO 2 dXAO 5 CAO 0 XAO PMME dt 1 KME W =V Kinetic and adsorption constants were calculated for each experiment, divided in series at constant temperature and series at constant catalyst concentration. The values obtained are shown in Table 4.

4. CONCLUSION We have shown that the esterication of diglycerol with oleic acid to yield diglycerol monooleate can be carried out selectively using Novozym 435 as the catalytic system. The selectivity obtained is >94% toward the desired product. The selective production of diglycerol monooleate can be well described as an irreversible second-order kinetic model, taking into consideration the adsorption of the reaction products in the
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Industrial & Engineering Chemistry Research catalyst with an average error minor than 3%. The estimated rate constant shows an Arrhenius dependence on temperature while the adsorption process shows a dependence on temperature which can be explained using the Vant Hos equation. The calculated activation energy and the adsorption enthalpy for the synthesis of diglycerol monooleate are 6.1 104 J 3 mol1 and 8.2 kJ 3 mol1, respectively. The values of these parameters were not available in the literature.

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AUTHOR INFORMATION
Corresponding Author

*Telephone: 34 91 3944175. E-mail: jam1@quim.ucm.es.

Notes

(9) Garca, E.; Ferrari, F.; Garca, T.; Martnez, M.; Aracil, J. A comparative study of the enzymatic esterication of diglycerol with fatty acids; 8th Mediterranean Congress of Chemical Engineering, Barcelona, Spain, 1999. (10) Garca, T.; Coteron, A.; Martnez, M.; Aracil, J. Esterication reactions catalyzed by immobilized lipases. Chem. Eng. Sci. 1996, 51, 2841. (11) Sanchez, N.; Coteron, A.; Martinez, M.; Aracil, J. Kineticanalysis and modelling of the esterication of oleic-acid and oleyl alcohol using cobalt chloride as catalyst. Ind. Eng. Chem. Res. 1992, 31, 1985. (12) Sanchez, N.; Martinez, M.; Aracil, J. Synthesis of oleyl oleate as a jojoba oil analog. J. Am. Oil Chem. Soc. 1992, 69, 1150. (13) Novo Nordisk Netherlands. Novozym 435. Novo Enzymes Technical Report Novo Industri A/S. Novo Alle (Denmark), 1995.

This material is available free of charge via the Internet at http://pubs.acs.org.

ACKNOWLEDGMENT This work has been funded by the Ministerio de Ciencia e Innovaci on from Spain (Project Plan Nacional of CTQ200909088). NOMENCLATURE CAO oleic acid concentration, mol 3 L1 (CAO)0 initial oleic acid concentration, mol 3 L1 CDGL diglycerol concentration, mol 3 L1 CME diglycerol monooleate ester concentration, mol 3 L1 Ea activation energy, J 3 mol1 (rAO) oleic acid reaction rate, mol 3 L1 3 min1 k kinetic reaction constant, L 3 mol1 3 min1 3 gCAT1 k0 pre-exponential factor, L 3 mol1 3 min1 3 gCAT1 KME adsorption constant, gCAT 3 gME1 T absolute temperature, K t time, min W catalyst weight, g oleic acid conversion XAO standard deviation REFERENCES
(1) Sanchez, N.; Martnez, N.; Aracil, J. Selective esterication of glycerine to 1-glycerol monooleate. 1. kinetic modelling. Ind. Eng. Chem. Res. 1997, 36, 1524. (2) Vicente, G.; Martnez y, M.; Aracil, J. Esteres Metlicos de Girasol: Alternativa al combustible Diesel Mineral. Ing. Qum. 1999, 153. (3) Akg ul, M.; Karabakan, A. Selective synthesis of monoolein with clinoptilolite. Microporous Mesoporous Mater. 2010, 131, 238. (4) Mouloungui, Z.; Rakotondrazafy, V.; Valentin, R.; Bachar, Z. Synthesis of glycerol 1-monooleate by condensation of oleic acid with glycidol catalyzed by anion-exchange resin in aqueous organic polymorphic system. Ind. Eng. Chem. Res. 2009, 48, 6949. (5) Mukesh, D.; Jadhav, S.; Sheth, D.; Banerji, A. A.; Thakkar, K.; Bevinakatti, H. S. Lipase-catalyzed esterication reactions. J. Chem. Technol. Biotechnol. 1997, 69, 179. (6) Kiriamiti, H. K.; Rascol, E.; Marty, A.; Condoret, J. S. Extraction rates of oil from high oleic sunower seeds with supercritical carbon dioxide. Chem. Eng. Process. 2001, 41, 711. (7) Charlemagne, D.; Legoy, M. D. Enzymatic synthesis of polyglycerol-fatty acid esters in a solvent-free system. J. Am. Oil Chem. Soc. 1995, 72, 61. (8) Babayan, V. K.; McIntyre, R. T. Preparation and properties of some polyglycerol esters of short and medium chain length fatty acids. J. Am. Oil Chem. Soc. 1968, 48, 307.
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