Process Biochemistry 44 (2009) 1068–1074

An integrated approach to produce biodiesel and monoglycerides by enzymaticinterestification of babassu oil (Orbinya sp)

Larissa Freitas, Patricia C.M. Da Ros, Julio C. Santos, Heizir F. de Castro *

Engineering School of Lorena, University of Sao Paulo, PO Box 116-12602-810, Lorena, SP, Brazil

A R T I C L E I N F O

Article history:

Received 30 July 2008

Received in revised form 14 May 2009

Accepted 19 May 2009

Keywords:

Biodiesel

Monoglycerides

Lipase

Silica–PVA composite

Glycerol

Babassu oil

A B S T R A C T

Two screenings of commercial lipases were performed to find a lipase with superior performance for the

integrated production of biodiesel and monoglycerides. The first screening was carried out under

alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding

ethyl esters (biodiesel). The second screening was performed under glycerolysis conditions to yield

monoglycerides (MG). All lipases were immobilized on silica–PVA composite by covalent immobiliza-

tion. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free

systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most

suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface

Methodology (RSM) was used to determine the optima operating conditions for each biotransformation.

For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction

at 39 8C using an oil-to-ethanol molar ratio of 1:7. For MG production, optima conditions corresponded

to oil-to-glycerol molar ratio of 1:15 at 55 8C, yielding 25 wt.% MG in 6 h reaction. These results show the

potential of B. cepacia lipase to catalyze both reactions and the feasibility to consider an integrated

approach for biodiesel and MG production.

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1. Introduction

Of the 80 million metric ton of fats and oils produced each yearworldwide, most are used directly in food, but about 2 million tonscan be used as raw material to produce a wide variety compounds ofindustrial applications [1]. Recently, great interest has appeared inthe biotechnological transformation of oils and fats, seeking the useof theserawmaterials intheproductionofhighvalue addedproductswith potential to be used in pharmaceutical industry, foods, plastics,renewable fuels, emulsifiers and other products [2–4].

Among the most promising processes for lipids modification arethe hydrolysis, synthesis of esters and transesterification of thesematerials in the presence of lipases–glycerol ester hydrolases; E.C.3.1.1.3 [5,6]. Lipases belong to the class of serine hydrolases andtherefore do not require any cofactor. The natural substrates oflipases are triacylglycerols, having very low solubility in water.Under natural conditions, they catalyze the hydrolysis of esterbonds at the interface between an insoluble substrate phase andthe aqueous phase in which the enzyme is dissolved. However, thepotential application of lipases in this segment is not exploitedenough. In this way, the reaction engineering is a critical issue that

* Corresponding author. Fax: +55 12 31595051.

E-mail address: heizir@dequi.eel.usp.br (H.F. de Castro).

1359-5113/$ – see front matter � 2009 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2009.05.011

needs to be solved prior to its industrial application. Many studieshave been performed to optimize the oil and fat modifications bylipases, including the use of strategies to improve the immobiliza-tion and stabilization of the enzyme, kinetic studies, modificationsand bioreactor development [1,6–9].

The present work makes use of lipases as catalysts in thebiotechnological transformation of oils and fats, aiming atproducing compounds with potential application in the generationof energy (biodiesel, monoalkyl esters) and in the food industry(monoglycerides, glyceryl esters). The fatty acid esters obtainedthrough the transesterification of oils with short chain alcohols(mainly methanol or ethanol) are known as biodiesel and showssimilar physical characteristics to petrol diesel that could be usedin motors of the cycle diesel without any modification [10,11]. Theglyceryl esters show relevant properties as emulsify agents and,depending on their composition can be used in creams, sauces andlotions formulations [12].

It is important to mention that in case biodiesel turns out to bean industrial reality, a surplus of approximately 25 thousand tonsof glycerol per year is expected to exist in the market (consideringdemand of 20 thousand ton/year), becoming strictly necessary todevelop new applications for low cost glycerol available frombiodiesel units operation [13,14]. The present work also looks forthis supply through the proposition of an enzymatic technology forusing this by-product to obtain high value added compounds.

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1069

This work was carried out in two steps. First of all, four selectedcommercially lipase preparations were screened to perform theproposed reactions using babassu oil and alcohols (ethanol orglycerol) in solvent free systems. All lipases were immobilized onsilica–PVA composite activated with epichlorohydrin following aprocedure previously described as suitable to immobilize lipasefrom several sources [4,15]. In the second step, conditions toperform both reactions using the most suitable lipase preparationwere optimized by using experimental design.

For transesterification reactions, ethanol was chosen as acylacceptor since it can easily be formed from renewable sources byfermentation, which makes the process of biodiesel production,totally independent from petrol derivatives. Therefore, ethanol ismore readily accepted for use in a variety of industrial situationsthan methanol, which has also as constraint its toxicity [4,6,11].

In relation to the lipid feedstock, babassu oil was selected,which is extracted from Orbinya martiana, a tree whose coconutscontain in average 7 wt.% of almonds with 62 wt.% of oil. Amongthem, lauric acid (C12H24O2) is the most important fatty acid[16,17]. On the pragmatic point of view, babassu cannot beconsidered as an oleaginous species, as it contains only 4% of oil.However, considering the millions of hectares of tropical forestswith a great amount of babassu palm trees and the possibilities ofthe integral usage of the coconut, babassu constitutes, potentiallyan extraordinary raw material source for biodiesel production,meanwhile the other parts of the tree can be used for otherpurposes [17,18]. In addition, it is more reasonable to use inedibleoil such as babassu oil, as edible oils are not in surplus supply.

2. Materials and methods

2.1. Materials

Lipase preparations manufactured from several companies include samples of

Candida antartica B (CAL B) kindly given by Novozymes (Araucaria, PR, Brazil);

Pseudomonas fluorescens (Lipase AK), Burkholderia cepacia (Lipase PS) and Penicillum

camembertii (Lipase G) bought from Amano Pharmaceuticals (Japan). All lipases

were used as received without further purification. Tetraethoxysilane was acquired

from Aldrich Chemical Co. (Milwaukee, WI, USA). Ethanol (minimum 99%) and

glycerol (purity, 99.5%, w/w) were purchased from Merck (Germany). Epichlor-

ohydrin, hydrochloric acid (minimum 36%), polyvinyl alcohol (MW 72,000) and

polyethylene glycol (molecular weight-1500) were supplied by Reagen (Rio de

Janeiro, RJ, Brazil). Babassu oil was kindly supplied by Cognis (Jacareı, SP, Brazil)

having the following composition in fatty acids: (w/v): 3.5% octanoic, 4.5% decanoic,

44.7% lauric, 17.5% myristic, 9.7% palmitic, 3.1% steriac, 15.2% oleic and 1.8% linoleic

with average molecular weight 709.90 g/mol. Solvents were of standard laboratory

grade (Synth, Sao Paulo, SP, Brazil). All the other reagents were of analytical degree.

2.2. Support synthesis and lipase immobilization

Silica–PVA composite was prepared by the hydrolysis and polycondensation of

tetraethoxysilane according to the methodology previously described [19]. Then,

the spheres were ground in the ball mill to attain nearly 0.175 mm diameter

particles, having the following properties: average pore diameter (22.91 A); surface

area BET (461.00 m2/g) and porous volume (0.275 cm3/g). Activation of silica–PVA

particles was carried out with epichlorohydrin at 2.5% (w/v) pH 7.0 for 1 h at room

temperature, followed by exhaustive washings with distilled water [13]. Activated

silica–PVA particles were soaked into hexane under stirring (100 rpm) for 1 h at

25 8C. Then, excess of hexane was removed and lipase was added at a ratio of 1:4

gram of enzyme per gram of support. PEG-1500 was added together with the

enzyme solution at a fixed amount (100 mL/g of support). Lipase-support system

was maintained in contact for 16 h at 4 8C under static conditions. The immobilized

lipase derivatives were filtered (nylon membrane 62HD from Scheiz Seidengaze-

fabrik AG, Thal Schweiz, Switzerland) and thoroughly rinsed with hexane.

2.3. Lipase activity

The immobilized derivatives were analyzed under both aqueous and non-

aqueous media. In the aqueous media, hydrolytic activities of immobilized lipase

derivatives were assayed by the olive oil emulsion method according to the

modification proposed by Soares et al. [20]. One unit (U) of enzyme activity was

defined as the amount of enzyme that liberates 1 mol of free fatty acid per min

under the assay conditions (37 8C, pH 7.0). Synthetic activities were carried out in a

closed spherical glass reactor containing 10 g of glycerol and lauric acid at 1:3 molar

ratio and incubating with immobilized lipase preparations (5% w/v based on the

total reaction volume). The esterification reaction was carried out at 60 8C with

continuous shaking at 200 rpm. The ester content was quantified by measurements

of the concentration of residual lauric acid in the reaction mixture. For these

measurements, withdrawn samples (150–200 mg) were diluted in 10 mL of an

ethanol/acetone 50:50 (v/v) mixture and titrated with KOH solution in ethanol

(0.05 mol/L).

2.4. Biodiesel synthesis

The reactions were performed in closed reactors with a capacity of 25 mL

containing 12 g of substrate consisting of babassu oil and anhydrous ethanol,

without the addition of solvents, at oil-to-ethanol molar ratio of 1:12. The mixtures

were incubated with the lipase from different sources immobilized on silica-PVA at

proportions of 20% (w/w) in relation to the total weight of reactants involved in the

reaction media [4]. The experiments were carried out at 40 8C. Reactions were

performed for a maximum period of 72 h under constant magnetic agitation of

150 rpm. For the time course studies, an aliquot of reaction medium was taken at

various time intervals and diluted in n-heptane for GC-analysis.

2.5. Glycerolysis reaction

The glycerolysis reactions were carried out in closed reactors with a capacity of

50 mL containing 25 g of substrate consisting of babassu oil and glycerol, without

the addition of solvents, at oil-to-glycerol molar ratio of 1:6. The enzyme amount

was kept constant at 10% (w/w) based on total mass of reagents. The temperature

was controlled at 45 8C. The reaction was mixed by magnetic stirrer at 200 rpm.

Samples taken at regular intervals were treated for extraction of the water and

glycerol followed previously methodology [3].

2.6. Experimental design

The influence of the variables (molar ratio and temperature) on both biodiesel

and monoglycerides formation were studied employing a 22 full factorial design,

with the addition of star points when necessary to compose a second order model.

Runs were performed at random. Three experiments were carried out at the center

point level, for experimental error estimation. Results were analyzed using

Statistica version 5 (StatSoft Inc., USA) and Design-Expert 6.0 (Stat-Ease

Corporation, USA) softwares. The statistical significance of the regression

coefficients was determined by Student’s test, the second order model equation

was evaluated by Fischer’s test and the proportion of variance explained by the

model obtained was given by the multiple coefficient of determination, R2.

2.7. Gas chromatography analysis

2.7.1. Biodiesel

Samples prepared as described above were analyzed by injecting 1 mL of heptane

solution and internal standard into a FID gas chromatograph Varian 3800 model

(Varian Inc. Corporate Headquarters, Palo Alto, CA, USA) equipped with flame-

ionization detector and with a 6 ft 5% DEGS on Chromosorb WHP, 80/10 mesh

column (Hewlett Packard, Palo Alto, CA, USA) following previous established

conditions [21]. All samples were measured in triplicate. The transesterification

yield was defined as the ratio between the produced and theoretical esters

concentrations � 100%. Theoretical ester concentrations were calculated by taking

into consideration the babassu oil fatty acid composition and its initial weight mass

in the reaction medium [4,21].

2.7.2. Monoglycerides

Mono–di and triacylglycerols were analyzed by gas chromatograph using a

Varian 3800 model (Varian, Inc., Corporate Headquarters, Palo Alto, CA, USA)

equipped with flame-ionization detector and with a 10 m � 0.25 mm � 0.12 mm CP

Sil 5CB capillary column (Varian Inc., Corporate Headquarters, Palo Alto, CA, USA).

The chromatograms were processed using a Varian data integrator version 4.51

computational program. Hydrogen was used as the carrier gas with a flow rate of

2 mL/min. The detector and injector temperatures were 350 8C. The column

temperature was set to 80 8C for 1 min and was then programmed at 20 8C/min to

320 8C which was maintained constant for 2 min. Other conditions were split ratio

of 1:20 and attenuation equal to 1. An organic phase was dissolved in hexane/ethyl

acetate (proportion of 1:1) which contained tetradecane as internal standard, and

the injection was carried out into the gas chromatograph.

3. Results and discussion

3.1. Screening of biocatalysts for biodiesel and monoglycerides

synthesis

The support obtained by the sol–gel technique was activatedwith epichlorohydrin and used to immobilize all tested lipases by

Table 1Hydrolytic and synthetic activities of the lipases immobilized on silica–PVA

composite.

Lipase source Hydrolytic

activity (U/g)

Synthetic

activity (U/g)

Pseudomonas fluorescens (lipase AK) 1210 � 68.36 26.32 � 1.34

Burkholderia cepacia (lipase PS) 1460 � 83.66 29.50 � 1.65

Candida antarctica B (Calb L) 48 � 10.40 27.33 � 1.31

Penicillium camembertii (lipase G) n.d. 39.83 � 2.01

Table 2Transesterification yield and productivity in the alcoholysis of babassu oil using

lipases from different sources immobilized on silica–PVA (data corresponding to

72 h reaction).

Lipase source Transesterification

yielda (%)

Productivity

(mg/g h)

Pseudomonas fluorescens (lipase AK) 70.28 5.4

Burkholderia cepacia (lipase PS) 90.93 7.0

Candida antarctica B (Calb L) 61.67 4.7

Penicillium camembertii (lipase G) 9.06 0.6

a The transesterification yield was defined as the ratio between the produced and

theoretical esters concentrations � 100. Theoretical esters concentration was

calculated by taking into consideration the total oil mass and its fatty acid

composition [4].

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–10741070

covalent binding and the catalytic activities in both aqueous andnon-aqueous media for the resulting derivatives are displayed inTable 1.

The highest hydrolytic activity was detected by the immobi-lized derivative obtained with B. cepacia (lipase PS) while thehighest synthetic activity was exhibited by the P. camembertii

(lipase G). Results also showed that the C. antarctica lipase (Calb L)derivative gave the lowest hydrolytic activity and the immobilizedP. camembertii lipase did not exhibit any hydrolytic activity at all.These variations can be attributed either to the different microbialsources of the tested lipases or to their distinct interactions withthe support. In relation to the lipase from P. camembertii, theabsence of the hydrolytic activity may be explained by itsclassification as mono- and di-acylglycerol lipases (partialglyceride hydrolases, EC 3.1.1.23), which have higher preferencetowards mono- and di-glycerides than triglycerides [22].

As the screening experiments were intended for an initialevaluation of the activity of the lipases, they were conducted undera preliminary set of reaction conditions that may not have been theoptimum set for all the lipases.

In a typical reaction for biodiesel production, 20% (w/w) of dryenzyme was added to the mixture of oil-to-ethanol molar ratio of1:12. The reactions were carried out at 40 8C according to thereaction setup as described in the materials and methods. Thescreening results for the tested lipases are shown in terms of totalethyl esters formation as a function of time (Fig. 1). Yields andproductivities attained at 72 h are displayed in Table 2.

The observation of the Fig. 1 and Table 2 indicates that all testedlipases were able to form ethyl esters from all fatty acids present in

Fig. 1. Profile of ethyl esters formation in the alcoholysis of babassu oil as a function

of time using lipases from different sources (*) lipase PS, (&) lipase AK; (~) lipase

Calb L and (~) lipase G immobilized on silica–PVA. All reactions were performed at

45 8C, using oil-to-ethanol molar ratio (1:12) under 150 rpm magnetic agitation.

the babassu oil. However, both reaction rate and yield weredependent on the lipase source tested. Ethyl esters concentrationsvaried in the range from 5.03 to 50.47 wt.%, corresponding totransesterification yields from 9.06 to 90.9% and productivitiesfrom 0.6 to 7 mg biodiesel/g h.

The best performance was attained by lipase PS rendering thehighest values for transesterification yield (90.9%) and productiv-ity (7 mg of biodiesel/g h). Lower performances were achieved bylipase AK (yield = 70.28%) and lipase Calb L (yield = 61.67%). LipaseG gave unsatisfactory results attaining less than 10% yield. Suchlow performance was associated to the stated preference towardsmono- and di-glycerides.

In all reaction systems, a good dispersion of the biocatalyst wasvisually observed in the substrate during the reaction. Inagreement with these results, among the tested lipases, lipasefrom B. cepacia (lipase PS) showed the highest activity towards thetransesterification of babassu oil with ethanol and was, therefore,selected for further study.

In relation to the monoglycerides (MG) synthesis, in a typicalreaction, 10% (w/w) of immobilized derivative was added to themixture of glycerol/babassu oil at a fixed molar ratio 6 to 1. Thescreening results for the tested lipases are shown in terms of MGconcentration (wt.%) as a function of time (Fig. 2).

Among the tested lipases, the best performance was attained bythe lipase PS which was able to form 9.7 wt.% of monoglycerideswithin 4 h reaction. Lipase AK gave slight lower performanceachieving 7.8% of MG within 6 h reaction. No satisfactory resultswere found for both Calb L and G lipase immobilized on silica–PVA,producing less than 2% MG. Such limitations were not verifiedwhen these enzymes were tested for monoglycerides formationusing the esterification route [3].

Based on these results, B. cepacia (lipase PS) was also chosen todetermine conditions at which monoglycerides formation wouldbe higher.

3.2. Experimental design for the lipase catalyzed biodiesel synthesis

Experimental design was used as a tool to study thecombined effect of oil-to-ethanol molar ratio and temperaturein the transesterification yield of the lipase catalyzed biodiesel.The range of molar ratio and temperature was between 1:7and 1:18 and 39–56 8C. In all experiments, the immobilizedPS derivative was used at proportions of 20% (w/w) in relationto the total weight of reactants involved in the reactionmedia.

The experimental matrix and the transesterification yields (%)are shown in Table 3. The results clearly showed that transester-ification yield was strongly affected by both variables. Thetransesterification yields varied from 71.43 to 98.44% and thehighest values was attained when lower levels of both molar ratio(1:7) and temperature 39 8C were used (run 1).

Fig. 2. Profile of monoglycerides formation in the glycerolysis of babassu oil using

lipases from different sources (*) lipase PS, (&) lipase AK; (~) lipase G and (~)

lipase Cal B immobilized on silica–PVA. All reactions were performed at 45 8C, using

oil-to-glycerol molar ratio (1:6) under 200 rpm magnetic agitation.

Table 4Estimated effects, standard errors and Student’s t test for transesterification yield in

biodiesel production using the 22 full factorial design.

Variables Effects Standard errors t p

Mean 81.54 �0.58 141.76 0.000*

Oil-to-ethanol molar ratio (x1) �6.66 �1.52 �4.37 0.048*

Temperature (x2) �10.64 �1.52 �6.99 0.020*

x1�x2 16.36 �1.52 10.75 0.009*

* Significant at 95% confidence level.

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1071

The statistical analysis of the results shows significant effect forboth variables and their interactions at 95% of confidence level(Table 4). Moreover, both variables had negative main effects.

The negative effect of molar ratio on the reaction yield can bedirectly related to this lipase source which appears to be lessethanol tolerant than the others described in the literature, such asPseudomonas fluorescens lipase [4] and Novozym 435 [23]. Forthese enzymes, reaction proceeds to a greater extent in thepresence of a large excess of ethanol. Therefore, for each lipasesource the excess of ethanol should be determined taking intoconsideration the complex mechanism involving reaction stoi-chiometry, polarity of the reaction medium and deactivation of theenzyme, among others.

In terms of the reaction stoichiometry, the use of equimolaramounts of ethanol to the number of fatty acids (FA) residues issufficient to obtain complete conversion of the FA residues to theircorresponding ethyl esters. However, some constraints maypreclude complete conversion. For example, in the immobilizedenzyme transesterification reaction, reactants initially form athree-phase system (triglyceride/alcohol/support). The reaction isdiffusion-controlled and poor diffusion between the phases exists.

Table 3Experimental design and results according to the 22 full factorial design to evaluate

the influence of the variables oil-to-ethanol molar ratio and temperature on the

transesterification yield (variables in coded values with real values in parenthesis)

for biodiesel production.

Runs Variables Transesterification

yield (%)a

Oil-to-ethanol molar ratio Temperature (8C)

1 �1 (1:7) �1 (39) 98.44

2 +1 (1:18) �1 (39) 75.42

3 �1 (1:7) +1 (56) 71.43

4 +1 (1:18) +1 (56) 81.14

5 0 (1:12.5) 0 (48) 83.20

6 0 (1:12.5) 0 (48) 80.60

7 0 (1:12.5) 0 (48) 80.53

a Correspondent to 48 h reaction.

As alkyl esters are formed, they act as a mutual solvent for thereactants and a two-phase liquid/solid system results [24]. As thereaction progresses towards completion and the by-product(glycerol) concentration increases, the alcohol and glycerol phaseseparate from the rich alkyl ester phase and a three-phase systemforms again. This is more likely to occur at lower initial alcoholconcentrations, and sometimes can result in an incompletereaction [25].

The entries in Table 3 indicate that for lipase PS highconversions of the oil to the FAEE (fatty acids ethyl esters) couldbe achieved when a lower excess of alcohol was utilized (runs 1and 3). Intermediate conversions were achieved at average molarratios (runs 5 and 6), but conversion was markedly decreasedwhen the largest excess of ethanol was present in the reactionmedium (runs 2 and 4). This decrease can be attributed toinactivation of lipase PS by ethanol. Watanabe et al. [26] havedemonstrated that high molar ratios of ethanol to FA residues leadto deactivation of lipases because of contact of the enzyme with theimmiscible polar organic phase formed as a consequence of a lackof complete solubility of the alcohol, as well as the productglycerol, in the oil phase. Thus, in the present work, alcohol inexcess was used to warrant the medium homogeneity during theprocess, but when this excess was increased even more, theinhibition effect in the enzyme was more important and the yielddecreased.

The influence of temperature on the transesterification yieldwas more important than that for oil/ethanol molar ratio, as seenclearly in Table 4. The negative influence of temperature is inagreement with thermal stability data for this immobilized lipasepreparation as previously determined by Da Ros [27]. According tothis study, lipase PS immobilized on silica–PVA composite is athermostable lipase preparation with a maximum activity in therange 50–60 8C. However, working temperatures in the range of40–50 8C are recommended to extend the operational stability.

The interaction effect of the variables was also statisticallysignificant at the same confidence level. As a result, at lower molarratio the transesterification yield was improved by lowering thetemperature (runs 1 and 3). But, at higher molar ratio thetransesterification yield was improved by rising the temperature(runs 2 and 4).

The main effects were fitted by multiple regression analysis to alinear model, and the best fitting response function can bedemonstrated by Eq. (1).

y ¼ 81:61� 3:33x1 � 5:32x2 þ 8:18x1 � x2 (1)

where y is transesterification yield (%) and x1 and x2 are molar ratioand temperature, respectively.

The statistical significance of this model was evaluated by the F-test (Table 5), which revealed that this regression is statisticallysignificant at a 95% probability level. The model did not show lackof fit and the determination coefficient (R2 = 0.99) indicates thatthe model can explain 99% of the variability.

A numeric optimization of the transesterification yield as afunction of the molar ratio and temperature was carried out using

Table 5Analysis of variance (ANOVA) for the regression of the model that represents

transesterification yield in biodiesel synthesis.

Source Sum of

squares

Degree of

freedom

Mean

square

F

Values Prob > F

Model 425.42 3 141.81 90.95 0.002*

Lack of fit 0.05 1 0.05 0.02 0.901

Pure error 4.63 2 2.32

Cor total 430.10 6

R2 0.99

* Significant at 95% confidence level.

Fig. 4. Ethyl esters profile in the alcoholysis of babassu oil using lipase from

Burkholderia cepacia (lipase PS) under the predicted conditions (oil-to-ethanol

molar ratio of 1:7 at 39 8C). Symbols: Ethyl esters from caprilic acid (x); capric acid

(*); lauric acid (~); myristic acid (!); palmitic acid (^); stearic acid (&); oleic

acid (5) and total esters (full line).

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–10741072

the software Design-Expert 6.0. According to this study, themaximum transesterification yield can be obtained at the lowestlevels of both variables (oil-to-ethanol molar ratio of 1:7 andtemperature of 39 8C) as displayed in the response surface (Fig. 3)correspondent to the model described by Eq. (1).

To confirm this model, experiments were carried out under theestablished condition and the differences between the experi-mental and theoretical values were lower than 1%. The runconducted to study particular conditions arising from the results ofthe experimental design attained a concentration of 77.46 wt.% ofethyl esters, which corresponded to a yield of 98%.

For this experiment, Fig. 4 displays the results considering thedifferent ethyl esters produced. No important changes, in the ethylesters concentration, after 48 h reaction was verified. The mainester produced was the ethyl laurate, followed by the ethylmyristate and ethyl oleate, with the other esters produced at loweramounts. This profile was as expected by taking into considerationthe babassu oil fatty acid composition [28] and was similar for allexperiments performed.

3.3. Experimental design for the lipase catalyzed monoglycerides

synthesis

The influence of the variables molar ratio and temperature inthe synthesis of monoglycerides by lipase catalyzed glycerolysis ofbabassu oil was, initially, evaluated in experiments carried outaccording to 22 full factorial design. Analysis of variance revealed a

Fig. 3. Response surface described by the model y1 that represents the

transesterification yield in the formation of biodiesel from babassu oil catalyzed

by PS lipase as a function of molar ratio (x1) and temperature (x2) according to

Eq. (1).

significant value for curvature (p < 0.05, data not shown),indicating the non-linearity of the model and thus justifying toadd points to the statistical design in order to determine amathematical model that provides the highest conversion of thestarting materials (glycerol and babassu oil) into monoglycerides.For this purpose, a central composite ‘‘22 + star’’ rotatable withthree replicates at the center points was built considering theworking range for oil-to-glycerol molar ratio from 1:8 to 1:22 andtemperature from 41 to 69 8C. In all experiments, the immobilizedPS derivative was used at proportions of 10% (w/w) in relation tothe total weight of reactants involved in the reaction media.

Table 6 shows the experimental matrix for the experimentaldesign together with data for the response variable (MG, wt.%).Results indicated that monoglycerides formation varied from 10 to28 wt.% of monoglycerides and the highest concentration was

Table 6Experimental design and results according to the central composite ‘‘22 + star’’

factorial design carried out to evaluate the influence of the variables oil-to-glycerol

molar ratio and temperature on the monoglyceride concentrations (MG) (variables

in coded values with real values in parenthesis) obtained in the glycerolysis of

babassu oil.

Runs Variables MG (wt.%)a

Oil-to-glycerol molar ratio Temperature ( 8C)

1 �1 (1:10) �1 (45) 16.48

2 +1 (1:20) �1 (45) 14.65

3 �1 (1:10) +1 (65) 17.24

4 +1 (1:20) +1 (65) 10.37

5 �1.41 (1:8) 0 (55) 13.84

6 +1.41 (1:22) 0 (55) 13.63

7 0 (1:15) �1.41 (41) 15.53

8 0 (1:15) +1.41 (69) 10.59

9 0 (1:15) 0 (55) 27.94

10 0 (1:15) 0 (55) 23.07

11 0 (1:15) 0 (55) 20.77

a Correspondent to 6 h reaction.

Table 7Estimated effects, standard errors and Student’s t test for monoglycerides formation

according to the central composite ‘‘22 + star’’ factorial design.

Variables Effects Standard

errors

t p

Mean 23.93 �1.64 14.57 0.000

Oil-to-glycerol molar ratio (x1) L �2.25 �2.01 �1.12 0.314

Oil-to-glycerol molar ratio (x1) Q �9.56 �2.40 �3.99 0.010*

Temperature (x2) L �2.63 �2.01 �1.31 0.248

Temperature (x2) Q �10.22 �2.40 �4.27 0.008*

x1�x2 �2.52 �2.85 �0.89 0.416

* Significant at 95% confidence level.

Table 8Analysis of variance (ANOVA) for the regression of the model that represents the

monoglycerides formation (wt.%) in the glycerolysis of babassu oil as a function of

oil-to-glycerol molar ratio (x1) and temperature (x2).

Source Sum of

squares

Degree of

freedom

Mean

square

F

Values Prob > F

Model 213.67 2 106.83 12.09 0.004*

Lack of fit 44.22 6 7.37 0.549 0.759

Pure error 26.81 2 13.40

Cor total 284.38 10

R2 0.75

* Significant at 95% confidence level.

Fig. 5. Response surface described by the model y1 that represents the glycerolysis

of babassu oil in the formation of monoglycerides (wt.%) catalyzed by PS lipase as a

function of molar ratio (x1) and temperature (x2) according to Eq. (2).

Fig. 6. Glycerides profile in the glycerolysis of babassu oil using lipase from

Burkholderia cepacia (lipase PS) under the predicted conditions (oil-to-glycerol

molar ratio of 1:15 at 55 8C). Symbols: monoglycerides (*); diglycerides (~);

triglycerides (&).

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1073

achieved using both variables at center point (molar ratio oil toglycerol of 1:15 and temperature at 55 8C).

In agreement with the test t of Student’s (Table 7) it was verifiedthat the quadratic effect of molar ratio (x1) and the temperature(x2) presented a significant influence (p < 0.05) on the mono-glycerides production. The linear effects and the interaction of thevariables were not statistically significant at the same confidencelevel and therefore were excluded from the model (Table 8).

From these results, the main effects were fitted by multipleregression analysis to a quadratic model, and the best fittingresponse function can be described by Eq. (2).

y2 ¼ 23:92� 4:77x21 � 5:11x2

2 (2)

where y2 is the response variable (monoglycerides, wt.%) and x1

and x2 represent the values coded for oil-to-glycerol molar ratioand temperature, respectively.

The statistical analysis of the model indicated that theregression is statistically significant (p = 0.04) at 95% confidencelevel, without significant lack of fit (p > 0.10). Moreover, the R2

value indicated that the model could explain more than 75% of theexperimental variability. Thus, the Eq. (2) was considered adequatefor describing the formation of monoglycerides as a function of thestudied variables and was used to plot the response surface asshown in Fig. 5.

The response surface described by the second order model forbabassu oil glycerolysis showed that the maximum monoglycer-ides formation could be attained at 55 8C and oil-to-glycerol molarratio of 1:15. These conditions correspond to runs 9–11 in Table 6and allow attaining 24 wt.% MG in 6 h. This result was similar todata described by Kaewtong et al. [12], using the same lipasesource immobilized on a different support (Accurel EP 100) andraw material (palm oil). In that work, 21% MG was obtained within24 h reaction.

The effect of oil-to-glycerol molar ratio can be explained byconsidering that alcohol in excess is needed to favor the MGaccumulation in the reaction medium, instead of diglycerides ortriglycerides [29]. However, high excess of glycerol can inhibit thelipase activity and affect, negatively, the process [30]. In the

present work, intermediary level of glycerol corresponded to thebest condition to lipase catalyzed MG production.

The same observation can be pointed for the temperature. Inthis process, low temperatures impair the homogeneity, restrain-ing the contact between the lipase and the hydrophobic substratesthat represents an obstacle to improve the MG yield. Thus, 55 8Cwas the optimum temperature value that could result in high MGproduction while preventing the lipase from thermal deactivationeffects (critical temperature at which the enzyme starts todeactivate was found to be 60 8C revealing a biocatalyst half-lifeof 6.24 h as reported by Da Ros [27].

Experiments were carried out under optima conditions andglyceride concentrations as a function of time are displayed inFig. 6. Under these conditions, about 25% of monoglycerides were

L. Freitas et al. / Process Biochemistry 44 (2009) 1068–10741074

accumulated in the purified reaction medium within 6 h reactionand this value remained almost constant up to 12 h process.Triglycerides were fast consumed in the first 3 h reaction withconsequent formation of diglycerides at concentrations reachinglevels of about 63%. Thus, the highest MG production wascorrespondent to 6 h reaction, having the following compositionin glyceryl esters: glyceryl laurate (57% of the total MG), glycerylmyristate (18% of the total MG), glyceryl oleate (11% of the totalMG) and others produced at lower amounts. Such profile was asexpected taking into consideration the babassu oil fatty acidcomposition, as already mentioned for biodiesel synthesis. Theseresults showed that the model fitted well with the experimentaldata, and thus described well the region studied.

4. Conclusion

Among four lipase sources, the enzyme from B. cepacia

immobilized on silica–PVA matrix was found to be the mostactive to catalyze both ethanolysis and glycerolysis of babassu oil.Further optimization was carried out which allowed to proposemathematical models representing each product formation in therange studied. For biodiesel production, the composed model was‘‘y = 81.61 � 3.33�x1 � 5.32�x2 + 8.18�x1�x2’’, where y is the transes-terification yield (%) and x1 and x2 are the coded values for molarratio and temperature, respectively. In this case, optima conditionswere: 39 8C using an oil-to-ethanol molar ratio of 1:7. For MGproduction, the composed model was ‘‘y2 = 23.92 � 4.77�x1

2 �5.11�x2

2’’, where y2 is the monoglycerides concentration (wt.%) andx1 and x2 represent the coded values for oil-to-glycerol molar ratioand temperature, respectively. In this case, the optima conditionswere 55 8C using an oil-to-glycerol molar ratio of 1:15. Theapproach proposed could allow associating environmental andeconomical concerns in addition to the use of non-edible vegetableoil as feedstock for biodiesel production and a by-product,‘‘glycerol’’, for production of high added value compound. Morework is still necessary in this fast-moving field.

Acknowledgements

The authors are grateful for the financial support provided byFAPESP (Fundacao de Amparo a Pesquisa do Estado de Sao Paulo)and CNPq (Conselho Nacional de Desenvolvimento Cientıfico eTecnologico), Brazil.

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