Biolubricants

1. An integrated approach to produce biodiesel and monoglycerides by enzymatic
interestification of babassu oil (Orbinya sp)
Larissa Freitas, Patricia C.M. Da Ro´s, Julio C. Santos, Heizir F. de Castro *
Engineering School of Lorena, University of Sa˜o Paulo, PO Box 116-12602-810, Lorena, SP, Brazil
1. Introduction
Of the 80 million metric ton of fats and oils produced each year
worldwide, most are used directly in food, but about 2 million tons
can be used as raw material to produce a wide variety compounds of
industrial applications [1]. Recently, great interest has appeared in
the biotechnological transformation of oils and fats, seeking the use
oftheserawmaterialsintheproductionofhighvalueaddedproducts
with 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 are
the hydrolysis, synthesis of esters and transesterification of these
materials in the presence of lipases–glycerol ester hydrolases; E.C.
3.1.1.3 [5,6]. Lipases belong to the class of serine hydrolases and
therefore do not require any cofactor. The natural substrates of
lipases are triacylglycerols, having very low solubility in water.
Under natural conditions, they catalyze the hydrolysis of ester
bonds at the interface between an insoluble substrate phase and
the aqueous phase in which the enzyme is dissolved. However, the
potential application of lipases in this segment is not exploited
enough. In this way, the reaction engineering is a critical issue that
needs to be solved prior to its industrial application. Many studies
have been performed to optimize the oil and fat modifications by
lipases, including the use of strategies to improve the immobiliza-
tion and stabilization of the enzyme, kinetic studies, modifications
and bioreactor development [1,6–9].
The present work makes use of lipases as catalysts in the
biotechnological transformation of oils and fats, aiming at
producing compounds with potential application in the generation
of energy (biodiesel, monoalkyl esters) and in the food industry
(monoglycerides, glyceryl esters). The fatty acid esters obtained
through the transesterification of oils with short chain alcohols
(mainly methanol or ethanol) are known as biodiesel and shows
similar physical characteristics to petrol diesel that could be used
in motors of the cycle diesel without any modification [10,11]. The
glyceryl esters show relevant properties as emulsify agents and,
depending on their composition can be used in creams, sauces and
lotions formulations [12].
It is important to mention that in case biodiesel turns out to be
an industrial reality, a surplus of approximately 25 thousand tons
of glycerol per year is expected to exist in the market (considering
demand of 20 thousand ton/year), becoming strictly necessary to
develop new applications for low cost glycerol available from
biodiesel units operation [13,14]. The present work also looks for
this supply through the proposition of an enzymatic technology for
using this by-product to obtain high value added compounds.
Process Biochemistry 44 (2009) 1068–1074
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.
ß 2009 Elsevier Ltd. All rights reserved.
* Corresponding author. Fax: +55 12 31595051.
E-mail address: heizir@dequi.eel.usp.br (H.F. de Castro).
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.05.011

2. This work was carried out in two steps. First of all, four selected
commercially lipase preparations were screened to perform the
proposed reactions using babassu oil and alcohols (ethanol or
glycerol) in solvent free systems. All lipases were immobilized on
silica–PVA composite activated with epichlorohydrin following a
procedure previously described as suitable to immobilize lipase
from several sources [4,15]. In the second step, conditions to
perform both reactions using the most suitable lipase preparation
were optimized by using experimental design.
For transesterification reactions, ethanol was chosen as acyl
acceptor since it can easily be formed from renewable sources by
fermentation, which makes the process of biodiesel production,
totally independent from petrol derivatives. Therefore, ethanol is
more readily accepted for use in a variety of industrial situations
than 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 coconuts
contain in average 7 wt.% of almonds with 62 wt.% of oil. Among
them, lauric acid (C12H24O2) is the most important fatty acid
[16,17]. On the pragmatic point of view, babassu cannot be
considered as an oleaginous species, as it contains only 4% of oil.
However, considering the millions of hectares of tropical forests
with a great amount of babassu palm trees and the possibilities of
the integral usage of the coconut, babassu constitutes, potentially
an extraordinary raw material source for biodiesel production,
meanwhile the other parts of the tree can be used for other
purposes [17,18]. In addition, it is more reasonable to use inedible
oil 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, Sa˜o 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 activated
with epichlorohydrin and used to immobilize all tested lipases by
L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1069

3. covalent binding and the catalytic activities in both aqueous and
non-aqueous media for the resulting derivatives are displayed in
Table 1.
The highest hydrolytic activity was detected by the immobi-
lized derivative obtained with B. cepacia (lipase PS) while the
highest 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 immobilized
P. camembertii lipase did not exhibit any hydrolytic activity at all.
These variations can be attributed either to the different microbial
sources of the tested lipases or to their distinct interactions with
the support. In relation to the lipase from P. camembertii, the
absence of the hydrolytic activity may be explained by its
classification as mono- and di-acylglycerol lipases (partial
glyceride hydrolases, EC 3.1.1.23), which have higher preference
towards mono- and di-glycerides than triglycerides [22].
As the screening experiments were intended for an initial
evaluation of the activity of the lipases, they were conducted under
a preliminary set of reaction conditions that may not have been the
optimum set for all the lipases.
In a typical reaction for biodiesel production, 20% (w/w) of dry
enzyme was added to the mixture of oil-to-ethanol molar ratio of
1:12. The reactions were carried out at 40 8C according to the
reaction setup as described in the materials and methods. The
screening results for the tested lipases are shown in terms of total
ethyl esters formation as a function of time (Fig. 1). Yields and
productivities attained at 72 h are displayed in Table 2.
The observation of the Fig. 1 and Table 2 indicates that all tested
lipases were able to form ethyl esters from all fatty acids present in
the babassu oil. However, both reaction rate and yield were
dependent on the lipase source tested. Ethyl esters concentrations
varied in the range from 5.03 to 50.47 wt.%, corresponding to
transesterification yields from 9.06 to 90.9% and productivities
from 0.6 to 7 mg biodiesel/g h.
The best performance was attained by lipase PS rendering the
highest values for transesterification yield (90.9%) and productiv-
ity (7 mg of biodiesel/g h). Lower performances were achieved by
lipase AK (yield = 70.28%) and lipase Calb L (yield = 61.67%). Lipase
G gave unsatisfactory results attaining less than 10% yield. Such
low performance was associated to the stated preference towards
mono- and di-glycerides.
In all reaction systems, a good dispersion of the biocatalyst was
visually observed in the substrate during the reaction. In
agreement with these results, among the tested lipases, lipase
from B. cepacia (lipase PS) showed the highest activity towards the
transesterification of babassu oil with ethanol and was, therefore,
selected for further study.
In relation to the monoglycerides (MG) synthesis, in a typical
reaction, 10% (w/w) of immobilized derivative was added to the
mixture of glycerol/babassu oil at a fixed molar ratio 6 to 1. The
screening results for the tested lipases are shown in terms of MG
concentration (wt.%) as a function of time (Fig. 2).
Among the tested lipases, the best performance was attained by
the lipase PS which was able to form 9.7 wt.% of monoglycerides
within 4 h reaction. Lipase AK gave slight lower performance
achieving 7.8% of MG within 6 h reaction. No satisfactory results
were found for both Calb L and G lipase immobilized on silica–PVA,
producing less than 2% MG. Such limitations were not verified
when these enzymes were tested for monoglycerides formation
using the esterification route [3].
Based on these results, B. cepacia (lipase PS) was also chosen to
determine conditions at which monoglycerides formation would
be higher.
3.2. Experimental design for the lipase catalyzed biodiesel synthesis
Experimental design was used as a tool to study the
combined effect of oil-to-ethanol molar ratio and temperature
in the transesterification yield of the lipase catalyzed biodiesel.
The range of molar ratio and temperature was between 1:7
and 1:18 and 39–56 8C. In all experiments, the immobilized
PS derivative was used at proportions of 20% (w/w) in relation
to the total weight of reactants involved in the reaction
media.
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. The
transesterification yields varied from 71.43 to 98.44% and the
highest values was attained when lower levels of both molar ratio
(1:7) and temperature 39 8C were used (run 1).
Table 1
Hydrolytic 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 Æ 68.36 26.32 Æ 1.34
Burkholderia cepacia (lipase PS) 1460 Æ 83.66 29.50 Æ 1.65
Candida antarctica B (Calb L) 48 Æ 10.40 27.33 Æ 1.31
Penicillium camembertii (lipase G) n.d. 39.83 Æ 2.01
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.
Table 2
Transesterification 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

4. The statistical analysis of the results shows significant effect for
both 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 be
directly related to this lipase source which appears to be less
ethanol tolerant than the others described in the literature, such as
Pseudomonas fluorescens lipase [4] and Novozym 435 [23]. For
these enzymes, reaction proceeds to a greater extent in the
presence of a large excess of ethanol. Therefore, for each lipase
source the excess of ethanol should be determined taking into
consideration the complex mechanism involving reaction stoi-
chiometry, polarity of the reaction medium and deactivation of the
enzyme, among others.
In terms of the reaction stoichiometry, the use of equimolar
amounts of ethanol to the number of fatty acids (FA) residues is
sufficient to obtain complete conversion of the FA residues to their
corresponding ethyl esters. However, some constraints may
preclude complete conversion. For example, in the immobilized
enzyme transesterification reaction, reactants initially form a
three-phase system (triglyceride/alcohol/support). The reaction is
diffusion-controlled and poor diffusion between the phases exists.
As alkyl esters are formed, they act as a mutual solvent for the
reactants and a two-phase liquid/solid system results [24]. As the
reaction progresses towards completion and the by-product
(glycerol) concentration increases, the alcohol and glycerol phase
separate from the rich alkyl ester phase and a three-phase system
forms again. This is more likely to occur at lower initial alcohol
concentrations, and sometimes can result in an incomplete
reaction [25].
The entries in Table 3 indicate that for lipase PS high
conversions of the oil to the FAEE (fatty acids ethyl esters) could
be achieved when a lower excess of alcohol was utilized (runs 1
and 3). Intermediate conversions were achieved at average molar
ratios (runs 5 and 6), but conversion was markedly decreased
when the largest excess of ethanol was present in the reaction
medium (runs 2 and 4). This decrease can be attributed to
inactivation of lipase PS by ethanol. Watanabe et al. [26] have
demonstrated that high molar ratios of ethanol to FA residues lead
to deactivation of lipases because of contact of the enzyme with the
immiscible polar organic phase formed as a consequence of a lack
of complete solubility of the alcohol, as well as the product
glycerol, in the oil phase. Thus, in the present work, alcohol in
excess was used to warrant the medium homogeneity during the
process, but when this excess was increased even more, the
inhibition effect in the enzyme was more important and the yield
decreased.
The influence of temperature on the transesterification yield
was more important than that for oil/ethanol molar ratio, as seen
clearly in Table 4. The negative influence of temperature is in
agreement with thermal stability data for this immobilized lipase
preparation as previously determined by Da Ro´ s [27]. According to
this study, lipase PS immobilized on silica–PVA composite is a
thermostable lipase preparation with a maximum activity in the
range 50–60 8C. However, working temperatures in the range of
40–50 8C are recommended to extend the operational stability.
The interaction effect of the variables was also statistically
significant at the same confidence level. As a result, at lower molar
ratio the transesterification yield was improved by lowering the
temperature (runs 1 and 3). But, at higher molar ratio the
transesterification yield was improved by rising the temperature
(runs 2 and 4).
The main effects were fitted by multiple regression analysis to a
linear model, and the best fitting response function can be
demonstrated by Eq. (1).
ˆy ¼ 81:61 À 3:33x1 À 5:32x2 þ 8:18x1 Á x2 (1)
where ˆy is transesterification yield (%) and x1 and x2 are molar ratio
and temperature, respectively.
The statistical significance of this model was evaluated by the F-
test (Table 5), which revealed that this regression is statistically
significant at a 95% probability level. The model did not show lack
of fit and the determination coefficient (R2
= 0.99) indicates that
the model can explain 99% of the variability.
A numeric optimization of the transesterification yield as a
function of the molar ratio and temperature was carried out using
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 3
Experimental 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 À1 (1:7) À1 (39) 98.44
2 +1 (1:18) À1 (39) 75.42
3 À1 (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.
Table 4
Estimated 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 Æ0.58 141.76 0.000*
Oil-to-ethanol molar ratio (x1) À6.66 Æ1.52 À4.37 0.048*
Temperature (x2) À10.64 Æ1.52 À6.99 0.020*
x1Áx2 16.36 Æ1.52 10.75 0.009*
*
Significant at 95% confidence level.
L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1071

5. the software Design-Expert 6.0. According to this study, the
maximum transesterification yield can be obtained at the lowest
levels of both variables (oil-to-ethanol molar ratio of 1:7 and
temperature 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 the
established condition and the differences between the experi-
mental and theoretical values were lower than 1%. The run
conducted to study particular conditions arising from the results of
the experimental design attained a concentration of 77.46 wt.% of
ethyl esters, which corresponded to a yield of 98%.
For this experiment, Fig. 4 displays the results considering the
different ethyl esters produced. No important changes, in the ethyl
esters concentration, after 48 h reaction was verified. The main
ester produced was the ethyl laurate, followed by the ethyl
myristate and ethyl oleate, with the other esters produced at lower
amounts. This profile was as expected by taking into consideration
the babassu oil fatty acid composition [28] and was similar for all
experiments performed.
3.3. Experimental design for the lipase catalyzed monoglycerides
synthesis
The influence of the variables molar ratio and temperature in
the synthesis of monoglycerides by lipase catalyzed glycerolysis of
babassu oil was, initially, evaluated in experiments carried out
according to 22
full factorial design. Analysis of variance revealed a
significant value for curvature (p < 0.05, data not shown),
indicating the non-linearity of the model and thus justifying to
add points to the statistical design in order to determine a
mathematical model that provides the highest conversion of the
starting materials (glycerol and babassu oil) into monoglycerides.
For this purpose, a central composite ‘‘22
+ star’’ rotatable with
three replicates at the center points was built considering the
working range for oil-to-glycerol molar ratio from 1:8 to 1:22 and
temperature from 41 to 69 8C. In all experiments, the immobilized
PS derivative was used at proportions of 10% (w/w) in relation to
the total weight of reactants involved in the reaction media.
Table 6 shows the experimental matrix for the experimental
design together with data for the response variable (MG, wt.%).
Results indicated that monoglycerides formation varied from 10 to
28 wt.% of monoglycerides and the highest concentration was
Table 5
Analysis 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. 3. Response surface described by the model ˆy1 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).
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).
Table 6
Experimental 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 À1 (1:10) À1 (45) 16.48
2 +1 (1:20) À1 (45) 14.65
3 À1 (1:10) +1 (65) 17.24
4 +1 (1:20) +1 (65) 10.37
5 À1.41 (1:8) 0 (55) 13.84
6 +1.41 (1:22) 0 (55) 13.63
7 0 (1:15) À1.41 (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.
L. Freitas et al. / Process Biochemistry 44 (2009) 1068–10741072

6. achieved using both variables at center point (molar ratio oil to
glycerol of 1:15 and temperature at 55 8C).
In agreement with the test t of Student’s (Table 7) it was verified
that 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 the
variables were not statistically significant at the same confidence
level and therefore were excluded from the model (Table 8).
From these results, the main effects were fitted by multiple
regression analysis to a quadratic model, and the best fitting
response function can be described by Eq. (2).
ˆy2 ¼ 23:92 À 4:77x2
1 À 5:11x2
2 (2)
where ˆy2 is the response variable (monoglycerides, wt.%) and x1
and x2 represent the values coded for oil-to-glycerol molar ratio
and temperature, respectively.
The statistical analysis of the model indicated that the
regression is statistically significant (p = 0.04) at 95% confidence
level, without significant lack of fit (p > 0.10). Moreover, the R2
value indicated that the model could explain more than 75% of the
experimental variability. Thus, the Eq. (2) was considered adequate
for describing the formation of monoglycerides as a function of the
studied variables and was used to plot the response surface as
shown in Fig. 5.
The response surface described by the second order model for
babassu oil glycerolysis showed that the maximum monoglycer-
ides formation could be attained at 55 8C and oil-to-glycerol molar
ratio of 1:15. These conditions correspond to runs 9–11 in Table 6
and allow attaining 24 wt.% MG in 6 h. This result was similar to
data described by Kaewtong et al. [12], using the same lipase
source immobilized on a different support (Accurel EP 100) and
raw material (palm oil). In that work, 21% MG was obtained within
24 h reaction.
The effect of oil-to-glycerol molar ratio can be explained by
considering that alcohol in excess is needed to favor the MG
accumulation in the reaction medium, instead of diglycerides or
triglycerides [29]. However, high excess of glycerol can inhibit the
lipase activity and affect, negatively, the process [30]. In the
present work, intermediary level of glycerol corresponded to the
best condition to lipase catalyzed MG production.
The same observation can be pointed for the temperature. In
this process, low temperatures impair the homogeneity, restrain-
ing the contact between the lipase and the hydrophobic substrates
that represents an obstacle to improve the MG yield. Thus, 55 8C
was the optimum temperature value that could result in high MG
production while preventing the lipase from thermal deactivation
effects (critical temperature at which the enzyme starts to
deactivate was found to be 60 8C revealing a biocatalyst half-life
of 6.24 h as reported by Da Ros [27].
Experiments were carried out under optima conditions and
glyceride concentrations as a function of time are displayed in
Fig. 6. Under these conditions, about 25% of monoglycerides were
Table 7
Estimated 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 Æ1.64 14.57 0.000
Oil-to-glycerol molar ratio (x1) L À2.25 Æ2.01 À1.12 0.314
Oil-to-glycerol molar ratio (x1) Q À9.56 Æ2.40 À3.99 0.010*
Temperature (x2) L À2.63 Æ2.01 À1.31 0.248
Temperature (x2) Q À10.22 Æ2.40 À4.27 0.008*
x1Áx2 À2.52 Æ2.85 À0.89 0.416
*
Significant at 95% confidence level.
Table 8
Analysis 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 ˆy1 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

7. accumulated in the purified reaction medium within 6 h reaction
and this value remained almost constant up to 12 h process.
Triglycerides were fast consumed in the first 3 h reaction with
consequent formation of diglycerides at concentrations reaching
levels of about 63%. Thus, the highest MG production was
correspondent to 6 h reaction, having the following composition
in glyceryl esters: glyceryl laurate (57% of the total MG), glyceryl
myristate (18% of the total MG), glyceryl oleate (11% of the total
MG) and others produced at lower amounts. Such profile was as
expected taking into consideration the babassu oil fatty acid
composition, as already mentioned for biodiesel synthesis. These
results showed that the model fitted well with the experimental
data, 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 most
active to catalyze both ethanolysis and glycerolysis of babassu oil.
Further optimization was carried out which allowed to propose
mathematical models representing each product formation in the
range studied. For biodiesel production, the composed model was
‘‘yˆ = 81.61 À 3.33Áx1 À 5.32Áx2 + 8.18Áx1Áx2’’, where yˆ is the transes-
terification yield (%) and x1 and x2 are the coded values for molar
ratio and temperature, respectively. In this case, optima conditions
were: 39 8C using an oil-to-ethanol molar ratio of 1:7. For MG
production, the composed model was ‘‘yˆ2 = 23.92 À 4.77Áx1
2
À
5.11Áx2
2
’’, where yˆ2 is the monoglycerides concentration (wt.%) and
x1 and x2 represent the coded values for oil-to-glycerol molar ratio
and temperature, respectively. In this case, the optima conditions
were 55 8C using an oil-to-glycerol molar ratio of 1:15. The
approach proposed could allow associating environmental and
economical concerns in addition to the use of non-edible vegetable
oil as feedstock for biodiesel production and a by-product,
‘‘glycerol’’, for production of high added value compound. More
work is still necessary in this fast-moving field.
Acknowledgements
The authors are grateful for the financial support provided by
FAPESP (Fundac¸a˜o de Amparo a` Pesquisa do Estado de Sa˜o Paulo)
and CNPq (Conselho Nacional de Desenvolvimento Cientı´fico e
Tecnolo´gico), Brazil.
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