Transesterification of babassu oil catalyzed by Burkholderia cepacia encapsulated in sol-gel matrix employing protic ionic liquid as an additive

Enzymatic transesterification from non-edible vegetable oil (babassu oil) and ethanol is provided. A set of seven experiments featuring a full 2 factorial design with three central points and different combinations of molar ratio and temperature as independent variables was employed. Transesterification reactions were catalyzed by Burkholderia cepacia lipase encapsulated in a hydrophobic matrix obtained by the sol-gel technique using protic ionic liquid (N-methylmonoethanolamine pentanoate) as additive and with conventional heating (40 – 56°C). Ethyl esters highest yield (51.90%) was obtained by experimental design with 1:7 molar ratio (oil:alcohol) and temperature at 40°C during 48h reaction. The process with a 5-fold increase of enzymatic load provided 98.69% ethyl esters yield with 4.29 mm s viscosity.


Introduction
Enzymatic transesterification has certain advantages over chemical transesterification, comprising easy recovery of glycerol, transesterification of glycerides with high free fatty acid contents, less energy intensive, which transforms the process for ethyl esters production into a cleaner process (BAJAJ et al., 2010).Moreover, the biocatalysts are eco-friendly (ANTCZAK et al., 2009).The steadily growing interest in lipases over the last two decades stems from their biotechnological versatility and the enzymes' ability to catalyze a broad spectrum of bioconversion reactions such as hydrolysis, esterifications, transesterifications, aminolysis and others (MINOVSKA et al., 2005).
Immobilization is an advantageous method that improves the stability of biocatalysts and provides for their repeated use and easy separation from the reaction medium (MINOVSKA et al., 2005;KATO et al., 2011).Lipases have been entrapped by sol-gel matrix employing different additives (KANDIMALLA et al., 2006;REETZ et al., 1996;SOUZA et al., 2012).
The use of additives in the immobilization process yields a significant improvement in the activity and stability of immobilized enzymes (REETZ et al., 1996;SOARES et al., 2003).Although specific literature has often reported the use of additives, such as albumin and polyethylene glycol (SOARES et al., 2003), sporopollenin (YILMAZ et al., 2010), zeolite (VIDINHA et al., 2006), calixarene derivatives (SAHIN et al., 2009), ionic liquid (SOUZA et al., 2013), the use of ionic liquids (IL) in all areas of chemical industries has an excellent prospective due to their unique properties (KESKIN et al., 2007).Ionic liquids are used as an additive in the immobilization process as agents that protect the enzyme from deactivation and modify the support´s morphology surface (SOUZA et al., 2013;FAUZI;AMIN, 2012).
Ionic liquids (ILs) are organic salts with melting points around or below the ambient temperature, negligible vapor pressure, high chemical and thermal stabilities, and constituted by organic cations and organic or inorganic anions (LIU et al., 2012).Protic ionic liquids (PILs) comprise a subset of the ionic liquids formed by the stoichiometric (equimolar) combination of a BrØnsted acid with a BrØnsted base and are characterized by their great ability to form H-bond and consequently show a strong interaction with polar solvents (ANOUTI et al., 2010).
The use of PILs in the process of enzyme immobilization is not usually reported in the literature.However, a study by Souza et al. (2013) using lipase Burkholderia cepacia encapsulated on hydrophobic matrixes obtained by sol-gel technique using protic ionic liquid as additive in the immobilization process showed promising results.The authors demonstrated an outstanding increase in the immobilization yield (over 1000%) compared to the activity offered by the immobilized enzymes without PIL.
Current research analyzes the effects of lipase from Burkholderia cepacia encapsulated in sol-gel matrix using protic ionic liquid in the transesterification of nonedible vegetable oil under conventional heating by employing a full factorial design 2 2 with three replicates of the center point.

Lipase immobilization
The sol-gel matrixes encapsulation methodology applied by Souza et al. ( 2013) was used with some modifications.It may be briefly described as follows: 30 mL of TEOS were dissolved in 36 mL of absolute ethanol under an inert nitrogen atmosphere.Further, 0.22 mL of hydrochloric acid was dissolved in 5 mL of slowly added ultra-pure water.The mixture was stirred (200 rpm) for 90 min.at 35°C.The commercial lipolytic enzyme from B. cepacia (2.7 g) was dissolved in a solution of 10 mL of ultra-pure water to which 1% (w v -1 ) of N-methylmonoethanolamine pentanoate (protic ionic liquid) was simultaneously added.Further, 1.0 mL of ammonium hydroxide dissolved in 6.0 mL of ethanol (hydrolysis solution) was added to the sol-gel reaction and the mixture was kept under static conditions for 24h until complete polycondensation.The bulk gel was washed with heptane and acetone and dried in a vacuum at room temperature for 72h.
The reloading of the previously immobilized system was carried by adsorption, following Freitas et al. ( 2009).The immobilized system was soaked in hexane and stirred (100 rpm) at 25°C.After 1h the excess of hexane was removed and lipase was added at a ratio of 1:4 gram of enzyme per gram of support.PEG-1500 and the enzyme solution were added together at a fixed amount (100 μL g -1 of support).Lipase-support system was maintained in contact for 16h at 4°C under static conditions, filtered and thoroughly rinsed with hexane.

Lipase activity
Enzymatic activities of free and immobilized lipase samples were assayed by olive oil emulsion method according to modifications by Soares et al. (1999).One unit (U) of enzyme activity was defined as the amount of enzyme that released 1 mol of free fatty acid per min under assay conditions (37°C; pH 7.0; 5 min.incubation).

Ethyl esters synthesis
The reactions were performed according to the methodology described previously by Freitas et al. (2009) in closed reactors with a capacity of 25 mL containing 12 g of substrate with babassu oil and anhydrous ethanol and without the addition of solvents.The mixtures were incubated with the immobilized lipase in the proportion of 20% (w w -1 ) in relation to the total weight of reactants involved in the reaction media.Reactions were performed for a maximum period of 48h under constant magnetic agitation of 150 rpm, stressing that two reactions entitled 1 and 2 were performed for 96h.An aliquot of reaction medium was taken at various time intervals for GC-analysis.
The ethyl esters formed were analyzed by gas chromatograph using a Varian CG 3800 model (Varian, Inc., Palo Alto CA USA) equipped with flame-ionization detector and 5% DEGS CHR-WHP 80/100 mesh 6 ft, 2.0 ID, in a stainlesssteel-packed column (Restek, Frankel Commerce of Analytic Instruments Ltd., São Paulo, São Paulo State, Brazil).Nitrogen constituted the gas carrier, with a flow rate of 25 mL min. - .Temperature programming was used.The column temperature was kept at 90°C for 3 min.,heated to 120°C at 25°C min. - and kept constant for 10 minutes.Further, temperature was programmed from 25°C min. - to 170°C and kept constant for 15 minutes.The temperatures of the injector and detector were set at 250°C.Data collection and analyses were performed with Galaxie Chromatography Data System 1.9.Calibration curves were built from standard ethyl esters using hexanol as internal standard.The transesterification yield was calculated by taking into account the mass of ester content detected by GC analysis and the total theoretical ester mass based on the molar ratio of the reaction.

Experimental design
Experiments were carried out according to the full 2 2 factorial design, with three replicates at the central point to study temperature effect, ethanol to babassu molar ratio (independents variables) on yield of ethyl esters.
Ethanol to babassu molar ratio (X 1 ) and temperature (X 2 ) were chosen as independent variables.Level selection was carried out from results obtained in a preliminary study (Table 1).The transesterification yield corresponded to response variable.Results were analyzed with Statistica 8.0 to verify the effect of the independent variable, assuming levels at p < 0.1 as statistical significance criterion.Student's test assayed the statistical significance of the regression coefficient.

Downstream procedure
At the end of the reaction with the highest yield on ethyl esters, the lipase was separated from the medium and washed three times with water to remove the free glycerol formed as a by-product.Residual ethanol was removed by pervaporation and the remaining water was removed by adding sodium sulfate salt to attain the final fatty acid ethyl ester product (ANDRADE et al., 2012).

Determination of viscosity
The absolute viscosity of biodiesel was measured with LVDV-II cone and plate spindle Brookfield viscosimeter (Brookfield Viscometers Ltd.UK) utilizing a CP 42 cone.A circulating water bath maintained temperature at 40°C for each analysis.Samples of 0.5 mL were used and measurements were replicated three times.Shear stress measurements were taken as a function of shear rate and the dynamic viscosity was determined as a slope constant.

Results and discussion
The lipase catalyzed reaction rate depends on the concentrations of enzyme, substrate, temperature and other factors (DA RÓS et al., 2012).The study of the molar ratio (X 1 ) and temperature (X 2 ) effect in the transesterification reaction was carried out in a solvent free system catalyzed by Burkholderia cepacia encapsulated in sol-gel matrix using protic ionic liquid (N-methylmonoethanolamine pentanoate) as additive (hydrolytic activity: 428.58 ± 25.79 U g -1 ).The enzymatic transesterification reaction is generally performed at lower temperature than that of the chemical reaction to prevent the loss of lipase activity (GOG et al., 2012).The temperature studied ranged between 40 and 56°C and the molar ratio oil: alcohol was 1:7 -1:13.
Results shown in Table 2 clearly indicated that the highest conversion babassu oil in ethyl esters, or rather, 51.90%, was obtained with the smallest excess alcohol (1:7) at 40°C for 48h reaction.Figure 2 shows that when molar ratio and temperature passed from +1 (1:13, 56°C) to -1 (1:7, 40°C), the highest conversions of babassu oil in ethyl esters were obtained.Table 3 shows that factors such as molar ratio, curvature and interaction between molar ratio and temperature were not significant at p > 0.1.Regression coefficient and p-values were calculated from the experimental results.Tables 3 and 4 show that temperature was the most significant independent variable.Thus, transesterification yields may be fitted to a three-dimensional surface described by a firstorder model as a function of X 1 and X 2 (Equation 1; Figure 2).Rates of R 2 (0.89) and R 2 adj (0.69) of the model suggest a close agreement between the experimental data and the theoretical values predicted by the model.No optimal conditions were observed in the response surface for the transesterification of babassu oil.Therefore, only the region corresponding to the best response may be identified.
The model equation for the response may therefore be written as (p < 0.1) in the following first-order equation that describes the response surface (1): Yield (%) = 41.60 -2.06*X 1 -6.39*X 2 + 1.86* X 1 *X 2 (1) In current assay, the amount of alcohol added in the babassu oil transesterification, equivalent to a molar ratio of 1:10 and 1:13, yielded ethyl esters lower than 50%.According to Tongboriboon et al. ( 2010), a greater amount of ethanol would cause an inhibitory effect on the enzymatic activity of the mixed lipase (Pseudomonas fluorescens and Candida rugosa).Alcohols are able to bind with proteins and induce their dehydration, resulting in a drastic loss of enzyme activity (CAMBON et al., 2009).Although theoretically transesterification reaction of one mole of triglyceride required three moles of ethanol, the yield of ethylic esters of fatty acids depended on the preferred equilibrium under various conditions (CHEIRSILP et al., 2008).Further, another hypothesis (CAMBON et al., 2009) demonstrated that when alcohols were dissolved in the oil phase, the effect of denaturation of the enzyme was greatly reduced due to the hydrogen bonds with the lipid species.It may therefore be suggested that the amount of alcohol in the reaction medium prepared at molar ratio oil to ethanol of 1:7 produced the highest transesterification yield possible by decreasing the substrate mass transfer limitations.
Moreover, temperature also influenced enzymatic activities.According to the Arrhenius equation, the reaction rate increased as temperatures rose.However, enzyme inactivation occurred and catalytic activity decreased above a certain temperature degree (SU et al., 2007).Cambon et al. (2009) reported that decreasing temperature from 55°C to 30°C increased the production of esters, with a growth in conversion rate from 55 to 73%.Freitas et al. ( 2009) achieved the highest transesterification yield while operating under the lowest excess ethanol (1:7) and temperature (39°C) when they used babassu oil as a triglycerides source.The authors used as catalyst the lipase from Burkholderia cepacia immobilized on SiO 2 -PVA and thus confirmed the lipase´s profile in this condition.The above is very similar to current study using Burkholderia cepacia lipase encapsulated in sol-gel matrix using protic ionic liquid (N-methylmonoethanolamine pentanoate) as an additive.
The performance of Burkholderia cepacia lipase encapsulated in current assay may be considered satisfactory when compared with performances reported by other lipase preparations, such as Burkholderia cepacia immobilized on niobium oxide hydrate (Nb 2 O 5 ) (DA RÓS et al., 2010), in which the immobilized biocatalyst provided 40.21% of the ethyl esters yield for a system composed of beef tallow and ethanol, for 48h.Lipase from immobilized Pseudomonas fluorescens, studied by Iso et al. (2001), showed a conversion of safflower oil between 0 and 50% for 50h when reaction consisting of the absence or 90 wt% of 1.4-dioxane.
Current assay showed promising results when compared to that by Souza et al. (2013).These researchers evaluated the production of ethyl esters from soybean oil and ethanol using lipase from Burkholderia cepacia encapsulated in matrix obtained by sol-gel technique and the same protic ionic liquid as additive.The authors reported 46.5% yield in 72h of reaction at 1:15.2 molar ratio and 0.075 g water.
Catalytic efficiency was the ratio between catalytic activity and enzyme loading.It provided the activity per milligram of immobilized enzyme.Therefore, this parameter was useful for comparative purposes among biocatalysts with different enzyme loading (SERRA et al., 2010).Rates obtained for hydrolytic activity (2176.5 U g -1 ± 99.48 U g -1 ) showed that catalytic efficiency of immobilized lipase by adsorption with enzyme loading increased more than five times than other enzyme loading studied (Burkholderia cepacia lipase encapsulated in sol-gel matrix using protic ionic liquid as additive).
In an attempt to evaluate the transesterification reaction by a biocatalyst with high lipase loading, a further experiment was performed and the results were compared with those attained by the biocatalyst with low lipase loading.Both reactions were carried out for 96h with babassu oil and ethanol as raw materials.Results may be analyzed under two different perspectives: the transesterification yield (%) and formation of ethyl monoesters as a function of time (Figures 3 and 4).The above results indicated that maximal transesterification yield (98.69%) was achieved by immobilized enzyme with high lipase loading (Reaction 2, Figure 4).Compared with reaction 1, the biocatalyst used in the experimental design exhibited a much lower transesterification yield (78.26%) (Figure 3).The second reaction revealed that total conversion of the fatty acids in the babassu oil reached their corresponding esters in the 96h reaction.The highest ethyl ester concentrations referred to ethyl laurate.In the two experiments approximately 30% of the C12 was formed consonant to the babassu oil fatty acids composition.
Comparatively, transesterification yield (%) in other studies with lipase immobilized on SiO 2 -PVA indicated similar results to those obtained in current research after enzyme loading increase (DA RÓS et al., 2010).Noureddini et al. (2005) also reported that when enzyme loading (Pseudomonas cepacia lipase) increased, there was a sudden surge in the formation of alkyl esters from the transesterification of soybean oil.
The difference between the percentages of conversion of babassu oil in the corresponding esters during the two reactions showed the influence of catalytic activity of the biocatalysts used.The viscosity values determined at 40°C were 6.67 and 4.29 mm² s -1 for reactions 1 and 2, respectively.According to the effective specified standards by the European Union EN-14214: (3.5 -5.0 mm² s -1 ), American Society for Testing and Materials ASTM D6751 (1.9 -6.0 mm² s -1 ) (BSG, 2012) and ANP 255 Brazil (2.5 -5.5 cSt) (DA RÓS et al., 2012), the viscosity rate attained in reaction 2 was within the recommended range.

Conclusion
Experimental design was employed to study ethyl esters production catalyzed by lipase from Burkholderia cepacia encapsulated in sol-gel matrix.A mathematical model was composed by the above technique which explained the variation of transesterification yields as a function of independent variables (molar ratio and temperature).The maximal transesterification yield was 51.9%, obtained in the molar ratio 1:7 and 40ºC.When the reaction was performed with biocatalyst with high lipase loading, the yield on ethyl esters increased to 98.69%, which in turn, originated a sample with appropriate property to be used as a fuel (viscosity value of 4.29 mm 2 s -1 ).

Figure 2 .
Figure 2. Response surface representing the transesterification yield of babassu oil catalyzed by lipase from Burkholderia cepacia encapsulated in sol-gel matrix using protic ionic liquid as additive.

Figure 3 .
Figure 3. Formation profile of ethyl monoesters as a function of time for reaction 1 (babassu oil transesterification with molar ratio 1:7 at 40°C) catalyzed by Burkholderia cepacia lipase encapsulated in sol-gel matrix using protic ionic liquid as additive (20 %, w w -1 ).

Figure 4 .
Figure 4. Formation profile of ethyl monoesters as a function of time for reaction 2 (babassu oil transesterification with molar ratio 1:7 at 40°C) catalyzed by Burkholderia cepacia lipase encapsulated in sol-gel matrix using protic ionic liquid as additive after enzyme loading increase (20 %, w w -1 ).

Table 1 .
Factor levels used according to the 2 2 full factorial design.

Table 2 .
Experimental design and values for yield conversion (%) according to the 2 2 full factorial design (variables in coded values with true values in parenthesis).

Table 3 .
Analysis of variance (ANOVA) for transesterification yield (%) of babassu oil using the 2 2 full factorial design.

Table 4 .
Regression coefficient, standard errors, and Student's t test for transesterification yield of babassu oil (%) using the 2 2 full factorial design.