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Biodiesel Synthesis by Enzymatic Transesterification of Palm Oil with Ethanol Using Lipases from Several Sources Immobilized on Silica–PVA Composite Ana B. R. Moreira,† Victor H. Perez,† Gisella M. Zanin,‡ and Heizir F. de Castro*,† Engineering School of Lorena, UniVersity of São Paulo, P.O. Box 116, 12602-810 Lorena, São Paulo, Brazil, and Department of Chemical Engineering, State UniVersity of Maringa, AV. Colombo 5790, E-46, 87020-900, Maringa-PR, Brazil ReceiVed July 11, 2007. ReVised Manuscript ReceiVed August 8, 2007
This work deals with the transesterification of palm oil with ethanol in a solvent free system using lipase from different sources (Thermomyces lanuginosus, Pseudomonas fluorescens, Burkholderia cepacia, Penicillium camembertii, and Candida antarctica, porcine pancreatic) immobilized on hybrid support polysiloxane–poly(vinyl alcohol). This is an exceptional option for the Brazilian biodiesel production, because both palm oil and ethanol are readily available in the country. The enzyme source showed strong influence on the transesterification yields, and the best performance was attained with the lipase from Pseudomonas fluorescens that reached almost full conversion (=98 %) in less than 24 h of reaction. The purified product (biodiesel) was straw yellow in color and essentially odorless. Purity of the fatty acid ethyl ester was found to be high having no glycerol bound as verified by NMR 13C (APT, attached proton test). In addition, the other properties such as low water content (0.02%), specific gravity (0.8), and viscosity (4.97 cSt) are in accordance with specifications recommended by the ASTM D6751 to be used as biofuel.
1. Introduction Esters from vegetable oils (biodiesel) are the best alternative for diesel as they do not demand any modification in the diesel engine, have a high energetic yield, and reduce both energy dependence on petroleum and air pollution.1,2 In addition, biodiesel is better than diesel fuel in terms of sulfur content, flash point, aromatic content, and biodegradability.2 Several types of vegetable oils (soybean, rapeseed, sunflower, and palm oils are the most studied), with a diversified compositions in fatty acids, can be used for the preparation of biodiesel. Among the raw materials with potential to obtain biodiesel, palm oil stands out for being the second most abundant oil in the world as well as for the palm being characterized as having superior productivity among all the other crops.3 Considering the type of alcohol, in Brazil it is advantageous to use anhydrous ethanol, which is already produced in large quantities to be mixed with gasoline.1 Biodiesel can be industrially produced by a chemical route using either acidic or alkaline catalysts, which give high conversion levels in a short reaction time. However, the conventional chemical route has several drawbacks: it is energy intensive, recovery of glycerol is difficult, the alkaline catalyst * To whom correspondence should be addressed. E-mail: heizir@ dequi.eel.usp.br. † University of São Paulo. ‡ State University of Maringa. (1) Pinto, A. C.; Guarieiro, L. L. N.; Michelle, J. C. R.; Ribeiro, N. M.; Torres, E. A.; Lopes, W. A.; Pereira, P. A. P.; Andrade, B. A. J. Braz. Chem. Soc. 2005, 16, 1313. (2) Knothe, G.; Van Gerpen, J.; Krahl, J. The Biodiesel Handbook; AOCS Press: Champaign, IL, 2005. (3) Kalam, M. A.; Masjuki, H. H. Biomass Bioenerg. 2002, 23, 471.
must be removed from the product, alkaline wastewater requires treatment, and free fatty acids and water interfere with the reaction.4 To minimize homogeneous process problems, attempts to use heterogeneous catalyst systems in alcoholysis of triglycerides have been made.5–8 These catalysts greatly simplify the posttreatment of the products (separation and purification). They can be easily separated from the system at the end of the reaction and may also be reused. Besides, the use of heterogeneous catalysts does not produce soaps through free fatty acid neutralization or triglyceride saponification. A large number of heterogeneous catalysts have been reported in the literature, including enzymes, zeolites, clays, guanidines heterogenized on organic polymers, ion-exchange resins, and oxides, among others.1 Although the enzymatic process is still not commercially developed, a number of articles have shown that enzyme holds promise as a catalyst. These studies consist mainly in optimizing the reaction conditions (temperature, alcohol/oil molar ratio, type of microorganism which generates the enzyme, enzyme amount, time, among others) to establish the characteristics for industrial applications.7–9 The key step in enzymatic processes lies in successful immobilization of the enzyme which will allow for its recovery (4) Al-Zuhair, S. Biotechnol. Prog. 2005, 21, 1442. (5) Granados, M. L.; Zafra Poves, M. D.; Martín Alonso, D.; Mariscal, R. F.; Cabello Galisteo, R.; Moreno-Tost, J. S.; Fierro, J. L. G. Appl. Catal., B 2007, 73, 317. (6) Kitakawa, N. S.; Honda, H.; Kuribayashi, H.; Toda, T.; Fukumura, T.; Yonemoto, T. Bioresour. Technol. 2007, 98, 416. (7) Salis, A.; Pinna, M.; Monduzzi, M.; Solinas, V. J. Biotechnol. 2005, 119, 291. (8) Noureddini, H.; Gao, X.; Philkana, R. S. Bioresour. Technol. 2005, 96, 769. (9) Paula, A. V.; Urioste, D.; Santos, J. C.; de Castro, H. F. J. Chem. Technol. Biotechnol. 2007, 82, 281.
10.1021/ef700399b CCC: $37.00 2007 American Chemical Society Published on Web 10/02/2007
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Table 1. Hydrolytic and Synthetic Activities of the Immobilized Lipases on POS-PVA Activated with Glutaraldehydea
source of lipase
hydrolytic activity (µmol/ (mg · min))
synthetic activity (µmol/ (g · min))
Thermomyces lanuginosus (Lipolase) Pseudomonas fluorescens (Lipase AK) Burkholderia cepacia (Lipase PS) Candida antarctica B (CALB L) Penicillium camembertii (Lipase G) porcine pancreatic lipase (PPL)
723 ( 7.1 765 ( 43.2 949 ( 54.4 48 ( 10.4 46 ( 8.3 276 ( 7.7
35.83 26.32 29.50 27.33 39.83 21.00
a Hydrolytic activity was determined by the olive oil emulsion method according to the modification proposed by Soares et al.10 One unit (U) of enzyme activity was defined as the amount of enzyme that liberates 1 µmol of free fatty acid per min under the assay conditions (37 °C, pH 7.0, 150 rpm). Synthetic activity was determined in the esterification of glycerol with lauric acid at a 1:3 molar ratio and incubating with immobilized lipase preparations according to methodology described by Langone and Sant′Anna.11
and reuse. In this work several lipase sources were immobilized by covalent binding on a sol–gel structure which was prepared by polycondensation of hydrolyzed tetraethoxysilane (TEOS) and poly(vinyl alcohol) (PVA). The immobilized derivatives were screened in the transesterification reaction of palm oil with ethanol followed by determining the effects of various reaction parameters on the reaction rate using the most active lipase source. The goal was to obtain full conversion of oil so that the results could be applied to the production of ethyl esters that fulfill its use as biofuel. This is an exceptional option for the Brazilian biodiesel production, because both palm oil and ethanol are readily available in the country. 2. Materials and Methods 2.1. Materials. Several crude lipase preparations manufactured from reputable companies include Candida antartica B (CALB L) and Thermomyces lanuginosus (Lipolase) from Novozymes (Araucária, PR, Brazil); Pseudomonas fluorescens (Lipase AK) and Burkholderia cepacia (Lipase PS) from Amano Pharmaceuticals (Japan) ;and porcine pancreatic lipase (Type II) from Sigma Co (U.S.A.). All lipases were used as received without further purification. TEOS was acquired from Aldrich Chemical Co. (Milwaukee, WI, U.S.A.). Glutaraldehyde (25% w/v), epichlorohydrin, hydrochloric acid (minimum 36%), ethanol (minimum 99%), PVA (molecular weight 72 000), and poly(ethylene glycol) (molecular weight 1500) were supplied by Reagen (RJ, Brazil). Palm oil was a kind gift from Agropalma (Para, Brazil) having the following composition in fatty acids (% w/v): 0.1% lauric, 1.2% mirystic, 46.8% palmitic, 3.8% stearic, 37.6 oleic, and 10.5% linoleic, with 849.0 g · mol-1 average molecular weight. All the other reagents were of analytical degree. 2.2. Support Synthesis and Lipase Immobilization. A polysiloxane–PVA (POS-PVA) hybrid composite was prepared by the hydrolysis and polycondensation of TEOS according to the methodology previously described.9 The resulting POS-PVA beads were ground in the ball mill to attain nearly 0.175 mm diameter particles and activated either glutaraldehyde or epichlorohydrin at 2.5% w/v pH 7.0 for 1 h at room temperature, followed by exhaustive washings with distilled water. Activated POS-PVA particles were used to immobilize all lipases tested in this study according to the procedure previously reported,9 attaining high retention of enzyme on the support (higher than 50%). The catalytic activities of the immobilized derivatives were analyzed under both aqueous and nonaqueous media following methodologies described by Soares et al.10 and Langone and Sant′Anna.11 (10) Soares, C. M. F.; de Castro, H. F.; Moraes, F. F.; Zanin, G. M. Appl. Biochem. Biotechnol. 1999, 77/79, 745.
2.3. Biodiesel Synthesis. The reactions were performed in closed reactors with a capacity of 25 mL containing 12 g of substrate consisting of palm oil and anhydrous ethanol, without the addition of solvents, at fixed molar ratio oil to alcohol (1:18). The mixtures were incubated with the lipase from different sources immobilized on POS-PVA at proportions of 20% w/w in relation to the total weight of reactants involved in the reaction media. The experiments were carried out at 40 °C. 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. Further studies were carried out with the selected lipase source to determine the influence of several parameters on the transesterification reaction, including lipase form (free or immobilized on POSPVA activated with glutaraldehyde or epichlorohydrin), ethanol/ oil molar ratio (9 and 18), and temperature (40 and 58 °C). Runs were carried out as described above. 2.4. Batch Operational Stability Tests. The operational stability of the immobilized system was assayed using immobilized lipase (1.0 g dry weight) and substrate containing palm oil and ethanol (molar ratio ) 18) in successive batches (40 °C/24 h/150 rpm). At the end of each batch, the immobilized lipase was removed from the reaction medium and washed with hexane, to remove any substrate or product eventually retained in the matrix. One hour later, the immobilized lipase was introduced in a fresh medium. Transesterification activity was estimated daily and expressed as micromoles of ethyl ester formed per minute per gram of catalyst. The biocatalyst half-life time (t1/2) was determined by applying the inverted linear decay model.12 2.5. Purification of Biodiesel. At the end of the reaction the lipase preparation was separated from the reaction medium, and the organic phase was twice washed with 1 vol of water to remove the remaining ethanol and free glycerol formed as a by-product. Residual water was removed by rotated evaporation, and the final fatty acid ethyl ester product was obtained. Water concentrations in the purified product were measured by coulometric Karl Fischer Titrometry using a Mettler DL 18 model (Mettler Scientific Co. Ltd.). Specific gravity was determined as recommended by ASTM1298. 2.6. Biodiesel Viscosity Determination. The absolute viscosity of biodiesel was determined with LVDV-II cone and plate spindle Brookfield viscometer (Brookfield Viscometers Ltd., U.K.) using a CP 42 cone. A circulating water bath was used to maintain the temperature at 25 or 40 °C ((0.1 °C) during the assays. The shear stress measurements were taken as a function of shear rate, and the dynamic viscosity was determined as a slope constant. Biodiesel samples of 0.5 mL were used, and the measurements were replicated three times. 2.7. GC Analysis. Samples prepared as described above were analyzed by injecting 1 µL of heptane solution and internal standard into a FID gas chromatograph (Varian CP 3800), using a 6 ft 5% DEGS on Chromosorb WHP, 80/10 mesh column (Hewlett Packard, Palo Alto, CA, U.S.A.) following previous established conditions.9 The yield was defined as the concentration ratio of transformed oil to initial oil × 100. All samples were measured in triplicate. 2.8. NMR 13C (APT, Attached Proton Test) Analysis. The palm oil and biodiesel were characterized by NMR 13C (APT) at 297 K using a Varian spectrometer, Mercury model, operating at 300 MHz. The standard reference used was tetramethylsilane (TMS) to adjust chemical displacements (δ ) 0) in a CDCl3 solvent.
3. Results and Discussions 3.1. Screening of Biocatalysts for Biodiesel Synthesis. Although lipases generally catalyze the hydrolysis of carboxylic esters, they bring about a range of bioconversion reactions such as esterification, transesterification, acidolysis, and aminolysis. (11) Langone, M. A. P.; Sant’Anna, G. L. Appl. Biochem. Biotechnol. 1999, 77–79, 759. (12) Pereira, E. B.; Zanin, G. M.; de Castro, H. F.; Braz, J. Chem. Eng. 2003, 20, 343.
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Figure 1. Profile for ethyl esters formation in the transesterification reaction of the palm oil with ethanol catalyzed by several immobilized lipases on POS-PVA. (a) Lipolase, (b) Lipase AK, (c) Lipase PS; (d) Lipase G; (e) Lipase CALB L, and (f) porcine pancreatic lipase (PPL). All reactions were performed at 40 °C, using a molar ratio of ethanol/oil (18) under 150 rpm magnetic agitation. Symbols: C12 (0); C14 (b); C16 (4); C18 (1); C18:1 (O); C18:2 (9); full line (total esters). Table 2. Yields and Productivities Attained in the Transesterification Reactions of Palm Oil with Ethanol Catalyzed by Lipases from Different Sources Immobilized on POS-PVA Activated with Glutaraldeyde lipases immobilized on POS-PVA Thermomyces lanuginosus (Lipolase) Pseudomonas fluorescens (Lipase AK) Burkholderia cepacia (Lipase PS) Candida antarctica B (CALB L) Penicillium camembertii (Lipase G) porcine pancreatic lipase (PPL)
transesterification productivity yield (%) (g/(L · h)) 55.00 91.00 75.10 10.10 7.10 18.60
3.51 6.40 4.83 0.66 0.47 1.20
Lipase screening was performed to find the lipase with the best catalytic activity in the transesterification of palm oil with ethanol. Six lipases (Lipolase, AK, PS, G, CALB L, and PPL) were screened for their transesterification activity. All lipases were previously immobilized on POS-PVA activated with glutaraldehyde. The resulting immobilized derivatives showed high catalytic activities in both aqueous and nonaqueous media as displayed in Table 1. To avoid reaction reversibility, special care has been taken to obtain immobilized preparations with low water level (90% reaction yield) activated with either glutaraldehyde or epichlorohydrin. Moreover, between the tested activating agents, the biocatalyst immobilized on POS-PVA activated with epichlorohydrin showed the highest activity and stability in the reaction medium, resulting in an increase of both reaction yield (99.4%) and productivity (8.16 g/(L · h)). These results are in agreement with the results reported by Iso et al.13 in which high transesterification activity (>90% conversion) was attained using Pseudomonas fluorescens lipase immobilized in a somewhat different support (kaolinite carrier). 3.3. Reusability of Immobilized Lipase in Transesterification. An important parameter in evaluating an immobilized enzyme is its lifetime or half-life for a particular reaction system. Moreover, long lipase lifespan in enzymatic reactions will significantly decrease the cost of the process, which will accelerate industrial applications of lipase technology. The stability of the immobilized system was also assessed by reusing the immobilized lipase five times in the synthesis of biodiesel. The synthesis was followed by evaluating the forma(13) Iso, M.; Chenb, B.; Eguchi, M.; Kudo, T.; Shrestha, S. J. Mol. Catal. B: Enzym. 2001, 16, 53.
Figure 5. Rheological curves for palm oil (0) and biodiesel (b) at 40 °C.
tion of ethyl esters per gram of biocatalyst per minute (transesterification activity). Figure 3 shows the residual activity as a function of operational time from which a slow decrease in the transesterification activity can be observed with a total reduction of 25% at the end of the fifth recycle, revealing a half-life time (t1/2) of 163 h. 3.4. Effect of the Ethanol/Oil Molar Ratio and Temperature on the Transesterification Yield. The effect of ethanol/oil molar ratio (9 and 18) and temperature (40 and 58 °C) on the transesterification reaction was also examined, and data is displayed in Table 3. According to these results, both temperature and higher concentration of ethanol enhanced the lipase AK transesterification activity. At a fixed molar ratio ethanol/oil of 18, raising the temperature incubation from 40 to 58 °C gave a positive effect on the process, resulting in an increase of the reaction rate. Therefore, reaction attained almost full conversion (97.95%) in 24 h, which corresponded to 23.87 g/(L · h) productivity.
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Figure 6. NMR 13C (APT): (a) palm oil and (b) biodiesel.
Decreasing the molar ratio from 18 to 9 also decreased the ethyl ester concentrations in the reaction medium. The formation of esters reached a maximum level (30 wt %) in a 24 h reaction, and this value was kept unchanged for the following 48 h. According to this data, the transesterification yield was almost two times lower than for reactions carried out with high molar ratio of reactants (ethanol/oil ) 18). This has led to the claim that the reaction rate might be limited by the concentration of the ethanol. Similar results were found by Noureddini et al.8 in the ethanolysis of soybean oil using starting materials at a molar ratio of ethanol/oil of 15.25 for which about 65% molar of ethyl esters was formed. Another important observation in this set of experiments may be related to the stability of the immobilized lipase AK in a medium with a high polarity. This means that this lipase source is less susceptible to the inhibition effects normally verified for lipases from other sources such as Candida rugosa and porcine
pancreatic lipase in highly polar reaction media as those constituted by ethanol.12,14 Taking in account these findings, an experiment using this immobilized derivative was carried out under the conditions established (reaction medium containing an ethanol/palm oil molar ratio of 18:1 at 58 °C) in a similar reactor configuration having a higher working capacity (100 mL) to monitor changes on the reaction medium over a period of 24 h. In this experiment, samples taken out from the reactor were analyzed in terms of ethyl esters and viscosity. The results are plotted in Figure 4 in which the variation of the viscosity and the transesterification yields are shown along with the reaction time. As can be (14) Urioste, D. Produção de biodiesel por catálise enzimática do óleo de babaçu com álcoois de cadeia curta. MSc Dissertation. School of Chemical Engineering of Lorena, FAENQUIL, Lorena-SP-Brazil, 2004 (available at www.hptt/faenquil.br, p 107).
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Table 4. Characteristics of the purified biodiesel from palm oil properties
specifications
viscosity at 40 °C (cSt) specific gravity at 25 °C water content (%) global yieldb (%)
1.9–6.0a 0.05a
biodiesel 4.97 0.80 0.02 96.5
a ASTM D6751 (United States): Standards Specification for Biodiesel (B100) Blend Stock for Distillate Fuels. b Formed ethyl ester with respect to the initial oil mass in the reaction medium, i.e., YGlobal ) [(biodiesel mass)/(oil mass)] × 100.
seen, a clear correlation between yield and viscosity was found in which the higher conversion the lower was the viscosity. Full conversion was found at 24 h for which the kinematic viscosity was 4.97 ( 0.03 cSt. 3.5. Rheological Properties. Because of the importance of the viscosity in the biodiesel evaluation, a rheological study of both palm oil and the transesterified product was carried out. Figure 5 shows typical curves for viscosity as a function of the shear rate for palm oil and biodiesel samples at 40 °C. Clearly, both curves exhibited Newtonian behavior; that is, the viscosity was constant and independent of the shear rate. Viscosity values were 28.72 ( 0.37 and 4.97 ( 0.03 cSt for palm oil and biodiesel, respectively. The latter is in accordance with both ASTM D5761 (United States) and EN 14214 (Europe) biodiesel standards that establish values in the ranges 1.9–6.0 cSt and 3.0–5.0 cSt, respectively. 3.6. Product Characterization. After the separation and purification steps, the product obtained was straw yellow in color and essentially odorless. Purity of the fatty acid ethyl ester was found to be high, having no glycerol bound as confirmed by the NMR 13C (APT). Spectra for palm oil and purified biodiesel are shown in Figures 6 a,b. The chemical displacement at 69.1 ppm registered in the spectrum for palm oil revealed the presence of the (CH) group of the glycerol in the triglyceride molecules which was confirmed by RMN 13C (APT). The absence of this peak in the spectrum for biodiesel supports the formation of the corresponding esters. The peak registered at =173ppm is characteristic for ester carbonyls; the chemical displacements in the range from 128 to 130 ppm corresponding to the insaturation present in the chains of the corre-
sponding fatty acid esters and the peak at 60.3 ppm are characteristic of the –CH2 of the ethoxi group. In addition, the other properties such as low water content (0.2%), specific gravity (0.8), and viscosity (4.97 cSt) may indicate that the product from this procedure can be used for applications such as direct diesel formulation (Table 4). Thus, the enzymatic transesterification process can be considered as an attractive option to the conventional chemical route. 4. Conclusions Enzymatic transesterification of triglycerides offers an environmentally more attractive option to the conventional physiochemical process. Transesterification of palm oil with ethanol was carried out by lipases from several sources, and among the tested enzymes the Pseudomonas fluorescens lipase had outstanding performance in relation to the transesterification activity, converting 90.98% of palm oil in the corresponding ethyl esters in 72 h which gave a productivity of 6.40 g of biodiesel/(L · h). The immobilized lipase AK was consistently more active and stable than the free lipase. Moreover, the biocatalyst activity was found to be also dependent on the support activating agent. Between the tested activating agents, epichlorohydrin was found to give the most stable and active immobilized derivative in the organic medium, resulting in enhanced transesterification yield (99.40%) and reaction productivity (8.16 g/(L · h)). Under the established operational conditions (58 °C and molar ratio of ethanol/oil ) 18), the immobilized Pseudomonas fluorescens lipase on POS-PVA activated with epichlorohydrin was found to be the most efficient biocatalyst to be used in the transesterification of the palm oil with ethanol, allowing almost total yield in ethyl ester to be achieved in 24 h, corresponding to a volumetric productivity of 23.87 g/(L · h). The purified product (biodiesel) showed viscosity values very close to those of the commercial diesel. Thus, the enzymatic transesterification process can be considered as an attractive option to the conventional chemical route. Acknowledgment. The authors acknowledge the financial assistance from CAPES, FAPESP, and CNPq (Brazilian Agencies). Thanks are also due to Dr. A.R. Rufino for the NMR 13C analysis. EF700399B
