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A R T I C L E I N F O A B S T R A C T
Article history: Two screenings of commercial lipases were performed to find a lipase with superior performance for the
Received 30 July 2008 integrated production of biodiesel and monoglycerides. The first screening was carried out under
Received in revised form 14 May 2009 alcoholysis conditions using ethanol as acyl acceptor to convert triglycerides to their corresponding
Accepted 19 May 2009
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-
Keywords: tion. The assays were performed using babassu oil and alcohols (ethanol or glycerol) in solvent free
Biodiesel
systems. For both substrates, lipase from Burkholderia cepacia (lipase PS) was found to be the most
Monoglycerides
Lipase
suitable enzyme to attain satisfactory yields. To further improve the process, the Response Surface
Silica–PVA composite Methodology (RSM) was used to determine the optima operating conditions for each biotransformation.
Glycerol For biodiesel production, the highest transesterification yield (>98%) was achieved within 48 h reaction
Babassu oil 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.
1359-5113/$ – see front matter ß 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2009.05.011
L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074 1069
This work was carried out in two steps. First of all, four selected 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
commercially lipase preparations were screened to perform the
continuous shaking at 200 rpm. The ester content was quantified by measurements
proposed reactions using babassu oil and alcohols (ethanol or of the concentration of residual lauric acid in the reaction mixture. For these
glycerol) in solvent free systems. All lipases were immobilized on measurements, withdrawn samples (150–200 mg) were diluted in 10 mL of an
silica–PVA composite activated with epichlorohydrin following a ethanol/acetone 50:50 (v/v) mixture and titrated with KOH solution in ethanol
procedure previously described as suitable to immobilize lipase (0.05 mol/L).
Table 1 Table 2
Hydrolytic and synthetic activities of the lipases immobilized on silica–PVA Transesterification yield and productivity in the alcoholysis of babassu oil using
composite. lipases from different sources immobilized on silica–PVA (data corresponding to
72 h reaction).
Lipase source Hydrolytic Synthetic
activity (U/g) activity (U/g) Lipase source Transesterification Productivity
yielda (%) (mg/g h)
Pseudomonas fluorescens (lipase AK) 1210 68.36 26.32 1.34
Burkholderia cepacia (lipase PS) 1460 83.66 29.50 1.65 Pseudomonas fluorescens (lipase AK) 70.28 5.4
Candida antarctica B (Calb L) 48 10.40 27.33 1.31 Burkholderia cepacia (lipase PS) 90.93 7.0
Penicillium camembertii (lipase G) n.d. 39.83 2.01 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
covalent binding and the catalytic activities in both aqueous and
theoretical esters concentrations 100. Theoretical esters concentration was
non-aqueous media for the resulting derivatives are displayed in calculated by taking into consideration the total oil mass and its fatty acid
Table 1. composition [4].
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 the babassu oil. However, both reaction rate and yield were
(lipase G). Results also showed that the C. antarctica lipase (Calb L) dependent on the lipase source tested. Ethyl esters concentrations
derivative gave the lowest hydrolytic activity and the immobilized varied in the range from 5.03 to 50.47 wt.%, corresponding to
P. camembertii lipase did not exhibit any hydrolytic activity at all. transesterification yields from 9.06 to 90.9% and productivities
These variations can be attributed either to the different microbial from 0.6 to 7 mg biodiesel/g h.
sources of the tested lipases or to their distinct interactions with The best performance was attained by lipase PS rendering the
the support. In relation to the lipase from P. camembertii, the highest values for transesterification yield (90.9%) and productiv-
absence of the hydrolytic activity may be explained by its ity (7 mg of biodiesel/g h). Lower performances were achieved by
classification as mono- and di-acylglycerol lipases (partial lipase AK (yield = 70.28%) and lipase Calb L (yield = 61.67%). Lipase
glyceride hydrolases, EC 3.1.1.23), which have higher preference G gave unsatisfactory results attaining less than 10% yield. Such
towards mono- and di-glycerides than triglycerides [22]. low performance was associated to the stated preference towards
As the screening experiments were intended for an initial mono- and di-glycerides.
evaluation of the activity of the lipases, they were conducted under In all reaction systems, a good dispersion of the biocatalyst was
a preliminary set of reaction conditions that may not have been the visually observed in the substrate during the reaction. In
optimum set for all the lipases. agreement with these results, among the tested lipases, lipase
In a typical reaction for biodiesel production, 20% (w/w) of dry from B. cepacia (lipase PS) showed the highest activity towards the
enzyme was added to the mixture of oil-to-ethanol molar ratio of transesterification of babassu oil with ethanol and was, therefore,
1:12. The reactions were carried out at 40 8C according to the selected for further study.
reaction setup as described in the materials and methods. The In relation to the monoglycerides (MG) synthesis, in a typical
screening results for the tested lipases are shown in terms of total reaction, 10% (w/w) of immobilized derivative was added to the
ethyl esters formation as a function of time (Fig. 1). Yields and mixture of glycerol/babassu oil at a fixed molar ratio 6 to 1. The
productivities attained at 72 h are displayed in Table 2. screening results for the tested lipases are shown in terms of MG
The observation of the Fig. 1 and Table 2 indicates that all tested concentration (wt.%) as a function of time (Fig. 2).
lipases were able to form ethyl esters from all fatty acids present in 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.
Table 4
Estimated effects, standard errors and Student’s t test for transesterification yield in
biodiesel production using the 22 full factorial design.
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
Fig. 2. Profile of monoglycerides formation in the glycerolysis of babassu oil using ratios (runs 5 and 6), but conversion was markedly decreased
lipases from different sources (*) lipase PS, (&) lipase AK; (~) lipase G and (~)
when the largest excess of ethanol was present in the reaction
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.
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
The statistical analysis of the results shows significant effect for to deactivation of lipases because of contact of the enzyme with the
both variables and their interactions at 95% of confidence level immiscible polar organic phase formed as a consequence of a lack
(Table 4). Moreover, both variables had negative main effects. of complete solubility of the alcohol, as well as the product
The negative effect of molar ratio on the reaction yield can be glycerol, in the oil phase. Thus, in the present work, alcohol in
directly related to this lipase source which appears to be less excess was used to warrant the medium homogeneity during the
ethanol tolerant than the others described in the literature, such as process, but when this excess was increased even more, the
Pseudomonas fluorescens lipase [4] and Novozym 435 [23]. For inhibition effect in the enzyme was more important and the yield
these enzymes, reaction proceeds to a greater extent in the decreased.
presence of a large excess of ethanol. Therefore, for each lipase The influence of temperature on the transesterification yield
source the excess of ethanol should be determined taking into was more important than that for oil/ethanol molar ratio, as seen
consideration the complex mechanism involving reaction stoi- clearly in Table 4. The negative influence of temperature is in
chiometry, polarity of the reaction medium and deactivation of the agreement with thermal stability data for this immobilized lipase
enzyme, among others. preparation as previously determined by Da Rós [27]. According to
In terms of the reaction stoichiometry, the use of equimolar this study, lipase PS immobilized on silica–PVA composite is a
amounts of ethanol to the number of fatty acids (FA) residues is thermostable lipase preparation with a maximum activity in the
sufficient to obtain complete conversion of the FA residues to their range 50–60 8C. However, working temperatures in the range of
corresponding ethyl esters. However, some constraints may 40–50 8C are recommended to extend the operational stability.
preclude complete conversion. For example, in the immobilized The interaction effect of the variables was also statistically
enzyme transesterification reaction, reactants initially form a significant at the same confidence level. As a result, at lower molar
three-phase system (triglyceride/alcohol/support). The reaction is ratio the transesterification yield was improved by lowering the
diffusion-controlled and poor diffusion between the phases exists. temperature (runs 1 and 3). But, at higher molar ratio the
transesterification yield was improved by rising the temperature
(runs 2 and 4).
Table 3
The main effects were fitted by multiple regression analysis to a
Experimental design and results according to the 22 full factorial design to evaluate linear model, and the best fitting response function can be
the influence of the variables oil-to-ethanol molar ratio and temperature on the demonstrated by Eq. (1).
transesterification yield (variables in coded values with real values in parenthesis)
for biodiesel production.
ŷ ¼ 81:61 3:33x1 5:32x2 þ 8:18x1 x2 (1)
Runs Variables Transesterification
yield (%)a where ŷ is transesterification yield (%) and x1 and x2 are molar ratio
Oil-to-ethanol molar ratio Temperature (8C)
and temperature, respectively.
1 1 (1:7) 1 (39) 98.44 The statistical significance of this model was evaluated by the F-
2 +1 (1:18) 1 (39) 75.42
3 1 (1:7) +1 (56) 71.43
test (Table 5), which revealed that this regression is statistically
4 +1 (1:18) +1 (56) 81.14 significant at a 95% probability level. The model did not show lack
5 0 (1:12.5) 0 (48) 83.20 of fit and the determination coefficient (R2 = 0.99) indicates that
6 0 (1:12.5) 0 (48) 80.60 the model can explain 99% of the variability.
7 0 (1:12.5) 0 (48) 80.53
A numeric optimization of the transesterification yield as a
a
Correspondent to 48 h reaction. function of the molar ratio and temperature was carried out using
1072 L. Freitas et al. / Process Biochemistry 44 (2009) 1068–1074
Table 5
Analysis of variance (ANOVA) for the regression of the model that represents
transesterification yield in biodiesel synthesis.
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.
Table 7
Estimated effects, standard errors and Student’s t test for monoglycerides formation
according to the central composite ‘‘22 + star’’ factorial design.
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).
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