Artículo_ie102560b.pdf

Published: April 27, 2011

r 2011 American Chemical Society 6609 dx.doi.org/10.1021/ie102560b | Ind. Eng. Chem. Res. 2011, 50, 6609–6614

ARTICLE

pubs.acs.org/IECR

Synthesis of Biosurfactants: Enzymatic Esterification of Diglyceroland Oleic Acid. 1. Kinetic ModelingMercedes Martínez, 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 inbiodegradable nonionic surfactant products. As an example of these, polyglycerol fatty acid esters have a wide field of application inmany industries as additives of cosmetics, food, photography, inks, etc. Also, the huge range of possible raw materials for theseproducts increases cited interest. The esterification of diglycerol with oleic acid using an enzyme (NOVOZYM-435) as catalyst wasstudied in this work. Several experiments were carried out at different temperatures, catalyst concentrations, and acid/alcohol molarratios. A pseudo-second-order kinetic model, including Langmuir adsorption factors for the acid, was developed. The results showthat this proposed kinetic model reproduces the experimental data with a maximum 10% error.

1. INTRODUCTION

Polyglycerol fatty acid esters have a wide field of application dueto their behavior as nonionic surfactants, biodegradability, hugerange of properties (liquids, solids, etc.), and the different rawmaterials available (vegetal, animal, saturated, unsaturated, etc.).The most important commercial products are the glycerol mono-stearate, monooleate, and monoricinoleate.1 The use of polygly-cerol fatty acid esters implies many advantages over using glycerolfatty acid esters. In this way, the use of polyglycerol fatty acid estersin the cosmetic industry leads a huge revolution due to the highwater adsorption of diglycerol, which helps to reduce the fattysensation of glycerol. In the food industry, the use of these esters asadditives in the E.U. is regulated in the European Directive 95/2/EC. These esters also have application in the lubricant (due to theirbiodegradability), polymer, and ink industries.

A growing interest in the biodiesel industry has arisen in thelast 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 mostinteresting in the near future.2

One of the critical factors for the viability of this process is thebenefit of the glycerol obtained as coproduct. Several works havebeen published on the esterification of glycerol and polyglycerol withfatty acids, looking for selective processes with high region-selectivityin which the catalyst can be easily recovered and, therefore, theenergy consumption reduced. Akg€ul researched the synthesis ofglycerol with oleic acid using clinoptilolite as a heterogeneouscatalyst.3 Another approach to the problem uses anion exchangeresins to produce the condensation of glycidol and oleic acid.4

However, the most suitable exploitation for this coproduct isthe enzymatic esterification with fatty acids or the polymerizationand later enzymatic esterification with fatty acids in order toobtain glycerol and polyglycerol fatty acid esters.5 The high priceand the wide application range make this alternative the mostsuitable one in the Mediterranean European countries, wherehigh fatty acid productions are achieved thanks to the modifiedseeds of cereals such as soy and sunflower, that can produce oilwhich has oleic acid content greater than 80%.6 An example of

this fatty acid production is the new plantation of 30.000 ha ofsunflower seed genetically treated for the production of higholeic acid, located in Spain.

In this work, the esterification of diglycerol with oleic acid isproposed. Glycerol, obtained as coproduct in the biodieselproduction process, would be used for the production of thediglycerol. On the other hand, this alternative allows the devel-opment of a national raw material (high oleic acid), whichemphasizes the economical interest in the process.

The enzymatic esterification of glycerol and polyglycerol withfatty acids has been carried out using different lipases as catalystsin solvent free systems.7,8 When using oleic acid as reagent, thebest results in acid conversion and monoester selectivity havebeen achieved with the commercial lipase Novozym-435 ascatalyst.9 Another lipase, Lipozyme-IM, has been used as catalystin these processes with different results. However, to the best ofour knowledge, there is no information about the kinetics of theesterification reaction that would eventually become necessaryfor an industrial production.

The objective of the present work is the study of the enzymaticesterification of diglycerol with oleic acid using Novozym-435and Lipozyme and IM as catalysts. The development of a kineticmodel10 and the process optimization, using the central compo-site design methodology,11,12 are also objectives in the secondpart of this research.

Calculations were performed using experimental data ob-tained under different catalyst concentrations, acid/alcoholmolar ratios, and temperatures.

2. EXPERIMENTAL SECTION

2.1. Raw Materials. Diglycerol of purity >98% (w/w)was supplied by Solvay Química S.L. (Spain), and oleic acid of

Received: December 22, 2010Accepted: April 27, 2011Revised: April 25, 2011

6610 dx.doi.org/10.1021/ie102560b |Ind. Eng. Chem. Res. 2011, 50, 6609–6614

Industrial & Engineering Chemistry Research ARTICLE

purity >98% (w/w) was supplied by Henkel Iberica. The cata-lysts used were immobilized thermostable lipases, Novozym,435and Lipozyme-IM, which were particularly useful for the synthe-sis of esters. This catalytic system was kindly supplied by NovoNordisk Bioindustry S.A.The main enzyme was a triacylglycerol hydrolase, E.C. 3.1.1.3,

which simultaneously acts as an effective carboxylesterase. Thepositional specificity of this system depends on the reactants. Insome reactions, it shows 1,3 positional specificity, whereas, inother reactions, the lipase functions as a nonspecific lipase. In themanufacture of Novozym 435, recombinant DNA technologywas used. The gene coding for the lipase was transferred from aselected strain of Candida antarctica to the host organismAspergillus oryzae. The enzyme obtained by this procedure wasimmobilized on a macroporous acrylic resin. The final productconsists of bead-shaped particles with a diameter of 0.3 mm. Thebulk 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 defined as 1 μmol oflauric acid converted to propyl laurate per minute per gram ofenzyme under standard conditions.13

Lipozyme IM is a nonspecific triacylglycerol lipase fromMucormiehei. The water content of this enzyme is 1�2 wt %/wt. Theactivity of the enzyme preparation was 60 batch interesterifica-tion units (BIU). One batch interesterification unit is defined as 1μmol of palmitic acid incorporated into triolein per minute atstandard conditions.The rest of the chemicals and solvents used for the chromato-

graphic analysis and in the recovery process of the catalyst weresupplied 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, and8 cm diameter. The reactor was equipped with stationary bafflesattached along the circumference. A marine-type propeller wasemployed. The impeller speed was set at 0.035 Np to maintainthe catalyst particles in good condition and because no significantinfluence of mass transfer on the process has been observedbetween 0.02 and 0.04 Np. A temperature recorder and con-troller 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 inthe range of temperature studied, without significant variations ofthe viscosity of the liquid phase or reaction volume. The reactor wasimmersed into a thermostatic bath capable of maintaining thereaction temperature within (0.1 �C of the set value by means ofan electrical device connected to a PID controller.2.3. Analytical Method. Reaction products were monitored

by gas chromatography/mass spectrometry (GC/MS) andquantitatively determined by capillary column gas chromatogra-phy. The GC/MS data were obtained on a 6890S Hewlett-Packard instrument. Gas chromatography was performed on afused-silica capillary column (OV-1, 12 m � 0.31 mm i.d., 0.17μm film). A Hewlett-Packard gas chromatograph was equippedfor slit-splitless injections (30 s). The GC/MS operating condi-tions were as follows: ionization energy, 70 eV; scan speed, 1100amu 3 s; mass range, 40�400 amu; data treated with a Hewlett-Packard 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 atthat temperature until all components had eluted. Quantitativegas chromatography analyses were performed on a Hewlett-Packard 5890 Series II instrument using the column and

conditions described above. A Hewlett-Packard 3396A integra-tor was connected to the chromatograph. The detector was anFID type at 270 �C, and the injection system was splitless. Thecarrier gas was helium, and the flow rate was 0.65 mL/min.Identification of the products of the reaction mixture was

performed by thin layer chromatography (TLC) using silica gelplates supplied by Merck. Products were separated with chloro-form/acetone (90:10 v/v) and visualized with sulphuric acid/water (10:90 v/v). Only oleic acid (Rf = 0.7) andmonoester (Rf =0.31) were detected while no glycerol trioleate was found in thesample.Several physicochemical characteristics of the produced sur-

factant (dyglicerol monooleate) have been determined. Skinirritation potential was measured by in vitro HET-CAM (hensegg test on the chorionallantoic membrane). Surface tensionwith determination of the CMC has been carried out using theDe No€uy method. Interfacial tension was determined by thespinning 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 thedesired temperature was reached, the catalyst was added, and thevacuum pump was turned on. The reactants were stirred during4 h. Samples were taken at regular intervals and at the end of thereaction, and analyzed by gas chromatography. During theexperiments, the following variables remained constant: acid/alcohol molar ratio, temperature, pressure/ and agitation speed.Before an experiment was started, the system was flushed withnitrogen.

3. RESULTS AND DISCUSSION

Thirteen experiments were carried out to determine the bestcatalyst and the influence of the temperature, catalyst concentra-tion, 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 selec-tivity versus time obtained for the different catalysts, setting thetemperature at 348.15 K, the catalyst concentration at 3% of thetotal substrate weight, and the diglycerol/oleic acid molar ratioat 1:1.After this study, Novozym-435 was chosen as the best catalyst

for this process because of the highest conversion (þ5%) andselectivity (8 times) obtained in comparison to the other catalysttested (Lipozyme-IM).

Table 1. Physicochemical Characteristics of the Surfactant

physicochemical characteristics

skin irritation slightly irritating

σ (mN/m) 27

γ (mN/m) 5.5

cmc (mmol/L) 0.12

Table 2. Experimental Conditions

operating temperature (K) 338.15 � 343.15 � 348.15

catalyst concentration (% wt) 0 � 1 � 3 � 5

diglycerol:oleic acid molar ratio (wt:wt) 3:1 � 1:1 � 1:3

stirring (Np) 0.035

pressure (Pa) 8.000



3.1.2. Influence of Stirring Speed. Preliminary experimentswere 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 at3%, and the diglycerol/oleic acid molar ratio at 1:1. At lowervalues of power number (high stirring speeds), no influence onthe initial reaction rate can be observed. This behavior indicatesthat at lower stirring speeds there is mass transfer dependencebecause of external diffusion mechanisms. However, no masstransfer limitation was detected at a power number below 0.04Np. 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 0.32and 0.8 mm. Figure 3 shows the initial reaction rate vs particlesize, using all the other variables at fixed values. The dependenceof acid conversion on particle diameter can be observed. Differ-ences between lower and upper sizes can produce almost anincrease of 20% on acid conversion. In fact, mass transfer

limitations take place, probably due to internal diffusion phe-nomena. However, mass transfer limitations can be neglected forcatalyst particle diameters below 0.55 mm. Therefore, all theexperiments were carried out using those particles found in thesieve 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 catalystconcentration at 3% and the diglycerol/oleic acid molar ratio at1:1. 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 themonoester selectivity. Maximum monoester selectivity wasachieved at the less operating temperature. This effect is due tothe lower mobility of the molecules and the higher energyrequired for the reaction at diglycerol tertiary carbons. The effectof the temperature was found to be similar for all catalystconcentrations and initial molar ratios.

Figure 2. Study of external diffusion. Influence of stirring speed.Catalyst conc = 3% of Novozym 435; T = 343.15 K; reactants molarratio = 1.

Figure 3. Study of internal diffusion. Influence of particle size. Catalystconc = 3% of Novozym 435; T = 343,15 K; reactants molar ratio = 1.

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

Figure 4. Influence of temperature on (a) acid conversion and (b) onmonoester selectivity.



3.2.2. Effect of the Initial Molar Ratio of Reactants. The effectof the reactants initial molar ratio was studied by setting thecatalyst 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 inthe monoester selectivity due to the excess of diglycerol present inthe reactor. The highermonoester selectivity was achievedwhen themolar ratio was set at 1:1. The acid conversion gap between thedifferent catalyst concentrations is practically the same; this impliesthat the oleic acid adsorption process in the catalyst is not significantat 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 thediglycerol/oleic acid initial molar ratio at 1:1 and the temperatureat 348.15 K. The results are shown in Figure 6, where the evolutionof both acid conversion and selectivity with reaction time can beobserved as a function of initial catalyst concentration.The influence, as expected, implies an increase in the reaction rate

andmonoester 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 bedescribed 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 triole-ate were detected.

oleic acidþ diglycerol f monooleateþ water

monooleateþ oleic acid f dioleateþ water

3.2.5. Effect of the Nonenzymatic Process. In order to estimatethe influence of the nonenzymatic process, an experiment

without catalyst was carried out at 348.15 K. The conclusionwas that no nonenzymatic reaction took place, obtaining practi-cally zero conversion to ester. This allowed us to take intoconsideration 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 isnecessary to estimate the dependence of the reaction rate on thecatalyst concentration. As is deduced from Figure 6a and shownin Figure 7, a linear relation between both parameters wasdetected. The evolution of the maximum reaction rate with theinitial concentration of immobilized enzyme was almost linearfor the three tested temperatures. This behavior made it possibleto consider the catalyst concentration as a linear parameter in thekinetic model.3.2.7. Irreversible Process Study. Assuming a second order

reaction (one for each reactant), the linearity between thereaction rate and the catalyst concentration, an irreversible

Figure 5. Influence of diglycerol/acid molar ratio (a) on acid conver-sion and (b) on monoester selectivity.

Figure 6. Influence of initial concentration of catalyst (a) on acidconversion and (b) on monoester selectivity.

Figure 7. Linear dependence of the kinetic model with catalyst con-centration at different temperatures.



process, and a negligible extension of the reversible reaction, thekinetic model proposed is as follows:

ð�rAOÞ ¼ � 1W

dCAO

dt¼ kðCAOÞðCDGLÞ ð1Þ

whereCAO =CDGL due to the 1:1 initial molar ratio between bothreactants.By using the integral method

XAO

1� XAO¼ kðCAOÞ0t ð2Þ

The kinetic parameters k and Ea were estimated using both theintegral method and the initial reaction rate method (differentialmethod). The reaction rate kinetic constant was estimated byusing a nonlinear regression method. The kinetic constant wascalculated for each experiment, divided in series at constanttemperature and in series at constant catalyst concentration. Thevalues 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 3mol

�1 and k0 = 1.6 � 107 L 3mol�13min�1

3 gCAT�1.

According to the experimental data, the model proposedappears to be consistent with the following expression:

ð�rAOÞ ¼ 1:6� 107� �

e�6:1�104=RTCAOCDGL ð3ÞFigure 8a shows the residual analysis of the process. As is

shown in this figure, a trend to negative residual values at largetime was detected.This effect could be due to an adsorption process of the

diglycerol monoester in the catalyst. In order to improve thekinetic model taking into consideration this adsorption process, adifferent model was proposed.3.2.8. Adsorption Kinetic Model. The proposed model was the

following

ð�rAOÞ ¼ kWC2AO

1þ KMECME

Ccat

ð4Þ

The adsorption constant was estimated using the kineticparameters, k, estimated before by using the initial reaction rateand a nonlinear regression method with the following expression(deduced from eq 4):

dXAO

dt

� �¼ kWðCAOÞ0ð1� XAOÞ2

1þ KMEðCAOÞ0XAOPMME

W=V

ð5Þ

Kinetic and adsorption constants were calculated for eachexperiment, divided in series at constant temperature and seriesat constant catalyst concentration. The values obtained areshown in Table 4.

Applying Van’t Hoff’s equation to the experimental resultsobtained, KMeo andΔHwere estimated to be KMEo = 9.8� 10�9

gCAT 3 gME�1 and ΔH = 8.2 kJ 3mol�1. Applying Arrhenius

equation to the results obtained using the initial reaction ratemethod, k0 and EA were estimated to be EA = 6.1� 104 J 3mol

�1

and k0 = 2.6 � 107 L 3mol�13min�1

3 gCAT�1.

According to the experimental data, the model proposedappears to be consistent with the following expression:

ð�rAOÞ ¼ 2:6� 107� �

e�6:1�104=RTWC2AO

1þ 9:8� 10�9� �

e�8:2�103=RT CME=Ccatð Þ ð6Þ

Figure 8b shows the residual analysis of the process. As isshown in this figure, no trend was detected. Also, the correlationcoefficients obtained show a good fit for the proposed model,with an average error minor than 3%.

4. CONCLUSION

We have shown that the esterification of diglycerol with oleicacid to yield diglycerol monooleate can be carried out selectivelyusing Novozym 435 as the catalytic system. The selectivityobtained is >94% toward the desired product.

The selective production of diglycerol monooleate can be welldescribed as an irreversible second-order kinetic model, takinginto consideration the adsorption of the reaction products in the

Table 3. Kinetic Constant Values: k (L 3mol�13min�1

3 gCAT�1)

integral method differential method

T (K) 103k 103σ 103k 103σ

338.15 4.6 1.8 6.5 3.2

343.15 5.7 2.8 7.9 5.2

348.15 8.3 4.8 13.1 8.2

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

Table 4. Adsorption Constant Values: KME (gCAT 3 gME�1)

T (K)\cat 1% 3% 5%

338.15 3.60 � 10�2 6.68 � 10�2 4.26 � 10�3

343.15 1.61 � 10�2 1.80 � 10�2 1.90 � 10�2

348.15 7.29 � 10�3 2.40 � 10�3 4.10 � 10�3



catalyst with an average error minor than 3%. The estimatedrate constant shows an Arrhenius dependence on temperaturewhile the adsorption process shows a dependence on tempera-ture which can be explained using the Van’t Hoff’s equation. Thecalculated activation energy and the adsorption enthalpy forthe synthesis of diglycerol monooleate are 6.1 � 104 J 3mol�1

and 8.2 kJ 3mol�1, respectively. The values of these parameters

were not available in the literature.

’AUTHOR INFORMATION

Corresponding Author*Telephone: þ34 91 3944175. E-mail: [email protected].

NotesThis material is available free of charge via the Internet at

http://pubs.acs.org.

’ACKNOWLEDGMENT

This work has been funded by the “Ministerio de Cienciae Innovaci�on” from Spain (Project Plan Nacional of CTQ2009-09088).

’NOMENCLATURECAO oleic acid concentration, mol 3 L

�1

(CAO)0 initial oleic acid concentration, mol 3 L�1

CDGL diglycerol concentration, mol 3 L�1

CME diglycerol monooleate ester concentration, mol 3 L�1

Ea activation energy, J 3mol�1

(�rAO) oleic acid reaction rate, mol 3 L�1

3min�1

k kinetic reaction constant, L 3mol�13min�1

3 gCAT�1

k0 pre-exponential factor, L 3mol�13min�1

3 gCAT�1

KME adsorption constant, gCAT 3 gME�1

T absolute temperature, Kt time, minW catalyst weight, gXAO oleic acid conversionσ standard deviation

’REFERENCES

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Girasol: Alternativa al combustible Diesel Mineral. Ing. Quím. 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 withglycidol catalyzed by anion-exchange resin in aqueous organic poly-morphic 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 esterification 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 sunflower seeds with supercritical carbondioxide. Chem. Eng. Process. 2001, 41, 711.(7) Charlemagne, D.; Legoy, M. D. Enzymatic synthesis of poly-

glycerol-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.

(9) García, E.; Ferrari, F.; García, T.; Martínez, M.; Aracil, J. Acomparative study of the enzymatic esterification of diglycerol with fattyacids; 8th Mediterranean Congress of Chemical Engineering, Barcelona,Spain, 1999.

(10) García, T.; Coteron, A.; Martínez, M.; Aracil, J. Esterificationreactions catalyzed by immobilized lipases. Chem. Eng. Sci. 1996,51, 2841.

(11) Sanchez, N.; Coteron, A.; Martinez, M.; Aracil, J. Kinetic-analysis andmodelling of the esterification of oleic-acid and oleyl alcoholusing cobalt chloride as catalyst. Ind. Eng. Chem. Res. 1992, 31, 1985.

(12) Sanchez, N.; Martinez, M.; Aracil, J. Synthesis of oleyl oleate asa jojoba oil analog. J. Am. Oil Chem. Soc. 1992, 69, 1150.

(13) Novo Nordisk Netherlands. “Novozym 435”. Novo EnzymesTechnical Report Novo Industri A/S. Novo Alle (Denmark), 1995.

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