RESEARCH PAPER
Selective ethanolysis of sunflower oil with Lipozyme RM IM, an immobilized Rhizomucor miehei lipase, to obtain a biodiesel-like biofuel, which avoids glycerol production through the monoglyceride formation Juan Calero1, Cristo´bal Verdugo2, Diego Luna1, Enrique D. Sancho3, Carlos Luna1, Alejandro Posadillo4, Felipa M. Bautista1 and Antonio A. Romero2 1
Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, 14014 Co´rdoba, Spain Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC, Avd. Las Palmeras, n84, 18100 Armilla, Granada, Spain 3 Department of Microbiology, University of Co´rdoba, Campus de Rabanales, Ed. Severo Ochoa, 14014 Co´rdoba, Spain 4 Seneca Green Catalyst S.A., Edif. Centauro, Parque Cientı´fico Tecnolo´gico de Co´rdoba, Rabanales 21, 14014 Co´rdoba, Spain 2
The obtaining of Ecodiesel, a biofuel applicable to diesel engines which keeps the glycerin as monoglyceride (MG), was achieved through a selective ethanolysis process of sunflower oil, by application of Lipozyme RM IM, a Rhizomucor miehei lipase immobilized on macroporous anion exchange resins. This biocatalyst that was already described in the synthesis of conventional biodiesel has also shown its efficiency in the present selective enzymatic process, after optimization of the influence of various reaction parameters. Thus, an adequate activity is obtained that is maintained throughout five successive reuses. Quantitative conversions of triglycerides (TG) with high yields to fatty acid ethyl esters (FAEE) were obtained under mild reaction conditions that correspond to the transformation of TG in a mixture of two moles of FAEE and a mole of MG, thus avoiding the glycerol production. Thus, the selective transesterification reaction of sunflower oil with absolute ethanol can be carried out under standard conditions with oil/ethanol volume ratio 12/3.5 (mL), at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, reaction temperature of 408C and 40 mg of Lipozyme RM IM. Under these experimental conditions six successive reactions can be efficiently carried out.
Introduction Nowadays the fossil fuel is the main primary source of energy. However, the worldwide demand for fuel is increasing (about 90 million barrels per day) and fossil fuel availability is becoming more limited; hence, it is becoming more accepted that the cheap and easily accessible fossil fuel era is coming to its end [1]. Thus, in the medium term obtaining alternative energies that are environmentally and economically viable [2] are essential. In the past years the biodiesel has been in this respect considerate as the most appropriate alternative to the fossil fuel. Thus, several methods have been reported for the biodiesel production from vegetable or waste cooking oils and animal fats [3]. In this respect, the vegetable Corresponding author:. Calero, J. ([email protected], [email protected])
oil has all the chemical–physical parameters similar to the fossil fuel; however, the viscosity is higher than petroleum diesel fuel in the order of 10–20 times. Thus, direct use of vegetable oil is not applicable to most of the actual diesel engines [4]. To reduce the viscosity of vegetable oil the methodology most accepted is the transesterification reaction for biodiesel production [5]. To obtain biodiesel an excess of methanol is normally used to shift the equilibrium to the production of fatty acid methyl esters (FAMEs). Along the three steps process, glycerol is obtained as a by-product. However, this glycerol is the main disadvantage of this method, not only because of reduction in the atomic yield of the process (approximately 10 per cent), but also the biodiesel obtained needs to be cleaned of the waste glycerol. In this cleaning process a lot of water is used [6].
www.elsevier.com/locate/nbt 1 Please cite this article in press as: Calero, J. et al., Selective ethanolysis of sunflower oil with Lipozyme RM IM, an immobilized Rhizomucor miehei lipase, to obtain a biodiesel-like biofuel, which avoids glycerol production through the monoglyceride formation, New Biotechnol. (2014), http://dx.doi.org/10.1016/j.nbt.2014.02.008
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To avoid the associated problems with the generation of glycerine, several alternative methods are in research to obtain glycerol derivatives, in the same transesterification reaction. These methodologies avoid the separation of glycerine, by incorporating some glycerol derivative in the reaction products [7]. Thus, mixtures of FAMEs and glycerol triacetate (triacetin) are products of the interesterification reaction of triglycerides with methyl acetate in the presence of strong acid catalysts, so that all these products could be used as components of new type of a patented novel biofuel, which strongly improves economy of the biofuel production. It is claimed that such a mixture named Gliperol exhibits fuel characteristics comparable to the traditional biodiesel [8]. The reaction between triglycerides and DMC produces a mixture of FAMEs and cyclic esters glycerol carbonate (GC) of fatty acid, FAGCs, which constitutes a novel biodiesel-like material, named DMC-BioD [9]. The interesterification reaction of triglycerides with dimethyl carbonate (DMC) can generate a mixture of FAMEs, FAGCs molecules and GC. By contrast, a protocol for the preparation of a new biodiesel that keeps glycerol into their composition as monoacylglycerols (MG) is developed via 1,3-regiospecific enzymatic transesterification of sunflower oil using lipases, free [10,11] and immobilized [12–14]. Thus, the already patented Ecodiesel is obtained through the 1,3-selective partial ethanolysis of the triglycerides with PPL (that stops the process in the second step), according to Fig. 3. In this way, a mixture of two parts of FAEE and one part of MG is obtained. This is a soluble product in the diesel fuel and exhibits furthermore better lubricity than conventional biodiesel [15–17]. However, the Ecodiesel term is currently ascribed to whichever blend of fatty acid alkyl ester with ethanol, alone or with any proportion of diesel fuel [18–21]. This biofuel named Ecodiesel is able to directly operate diesel engines, alone or in whichever mixture with diesel fuel, without any separation or purification process (Fig. 1). To improve the viability and competitiveness of the enzymatic process to obtain Ecodiesel, the present study aims to evaluate in this process the commercial biocatalyst Lipozyme RM IM, a Rhizomucor miehei lipase immobilized in macroporous anion exchange resins, which has already been described in the synthesis of conventional biodiesel [22] as well as in biodiesel-like biofuels by interesterification with DMC [23,24] or methyl acetate [24]. Thus, in this study we attempt to put in value the 1,3 selective behavior of this commercial immobilized R. miehei lipase as well as
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the possibility of its reuse, to make profitable the production of alternative biofuels that avoids the glycerol generation, using an enzymatic approach. In this regard, this study is conducted to evaluate the influence of various reaction parameters on the activity of this immobilized R. miehei lipase as well as the reuse possibilities, to overcome the existing limitations in the use of industrial lipases, mainly associated with their high costs.
Materials and methods Materials Commercial sunflower oil was obtained locally. The ethyl esters of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were from Accustandard, and the hexadecane (cetane) was obtained from Sigma–Aldrich and they all were chromatographically pure materials. Other chemicals like absolute ethanol and sodium hydroxide were obtained commercially from Panreac and they were pure analytical compounds (99%). The Lipozyme RM IM, a R. miehei lipase immobilized in beads from macroporous anion exchange resins, was kindly provided by Novozymes A/S.
Analytical method Reaction products were monitored by capillary column gas chromatography, using a Varian 430-GC gas chromatograph, connected to a HT5 capillary column (25 m 0.32 mm ID 0.1 mm, SGE, Supelco) with a flame ionization detector (FID) at 4508C and splitless injection at 3508C. As carrier gas helium is used, with a flow of 1.5 mL/min. A heating ramp from 908C to 2008C at a rate of 78C/min has been applied, followed by another ramp from 2008C to 3608C at a rate of 158C/min, maintaining the temperature of the oven at 3608C for 10 min. To determine the absence of glycerol as reaction product, the ramp was started from 458C. As internal standard n-hexadecane (cetane) is used to quantify the reaction conversion. The fatty acid ethyl esters (FAEE) and glycerides (monoglyceride, diglyceride and triglyceride – MG, DG and TG) are determined with the help of some commercial standard fatty acid esters. This method allows us to make a complete analysis of the sample in a single injection and in a time not higher than 60 min, which simplifies the process and increases the speed of analysis [10–14].
Kinematic viscosity measurements The viscosity is an important parameter for the correct running of diesel engines. In fact, the transesterification reaction is developed
FIGURE 1
New biodiesel-like biofuel (Ecodiesel) patented by University of Cordoba, obtained by lipase 1,3 selective ethanolysis of sunflower oil that integrated the glycerol as monoacylglycerols. It is formed by two moles of ethyl esters of fatty acids (FAEE) and one mole of monoglyceride (MG).
2
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Ethanolysis reactions To carry out the study the optimizations of operation conditions of Lipozyme RM IM, several parameters previously optimized with other lipases before being studied such as PPL and Lipopan 50 BG [10–14], have been maintained constant, to better study other variables more specifically dependent of every lipase, and that may produce important changes in the lipase activity, such as reaction temperature, lipase amount and mainly the water activity (aw). This latter one is very important to operate with waste oils, which usually contain variable amounts of water. Thus, a methodology OVAT (One Variable At Time) was used to study the influence of temperature, and it used enzyme loading and the water content in the reaction medium. These reactions were performed according to the experimental procedure previously described [10–14] to determine the optimal conditions for obtaining the selective ethanolysis reaction, such as amount of lipase, water amount and temperature operating at the same pH and oil/ethanol molar ratio (1/6). Thus, enzymatic assays are carried out with 9.4 g (12 mL, 0.01 mol) of commercial sunflower oil and 3.5 mL of absolute ethanol, at controlled temperatures (20–408C) in a 25 mL round bottom flask. Reaction mixtures were stirred with a conventional magnetic stirrer at a higher stirring speed than 300 rpm, to avoid mass transfer limitations, along a reaction time of 2 hours, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH. In this regard, a blank reaction in the presence of the highest quantity of solution of NaOH was performed to rule out a potential contribution from the homogeneous NaOH catalyzed reaction. Less than 10% conversion of the starting material was obtained, so that a homogenous base catalysis contribution can be considered as negligible under the investigated conditions. The different amounts of Lipozyme RM IM and commercial lipase are studied in the range of 0.05–0.1 g (0.1–1 wt%), temperatures between 30 and 708C and water
amounts from 0 to 0.3 wt%. Finally, a study of reuses is also carried out.
Results and discussion Influence of the reaction temperature To study the influence of the reaction temperature on the conversion values of the ethanolysis processes studied, a series of experiments are conducted with three different amounts of Lipozyme RM IM biocatalyst, in the range 20–100 mg, and at three different temperatures in the range 30–708C. Thus, these experiments are carried out under standard conditions, with an oil/ ethanol volume ratio 12/3.5 (mL) and constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH. The data obtained are shown in Fig. 2. From the results obtained a maximum activity at 408C for the three studied heterogeneous biocatalyst amounts can be easily seen. Similarly, a slight increase in the enzymatic activity can be seen when immobilized lipase amount increases from 20 to 50 mg, and then it falls drastically when 100 mg of Lipozyme is used. These results do not offer any doubt on the influence of temperature on the biocatalytic behavior of Lipozyme, clearly showing that the optimum operating temperature is 408C. However a more detailed assessment of the influence of the content of lipase is needed to determine the optimal biocatalyst amount to obtain maximum conversion per gram biocatalyst, that is, the specific enzymatic activity of Lipozyme in the selective ethanolysis process is studied.
Influence of the lipase amount To obtain more accurate information on the optimal value to optimize the conversion, the influence of the biocatalyst amount in the reaction yield is evaluated by increasing the lipase Lipozyme RM IM amount from 0.1 to 1 wt%, with respect to the oil weight and at the optimum value of temperature obtained. Thus, these experiments are carried out at 408C, with an oil/ethanol volume ratio 12/3.5 (mL) and constant pH obtained by the addition of
FIGURE 2
Influence of the reaction temperature on the conversion in the selective enzymatic ethanolysis of sunflower oil, using different amounts of Lipozyme RM IM lipase, operating under standard reaction conditions.
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Research Paper
to obtain a noticeable reducing in the viscosity, thus this biofuel can be used in the diesel engines. Thus, accurate viscosity measurements are crucial to assess the quality of biofuels produced; therefore, the characterization of this parameter is essential to evaluate the result obtained in the process of ethanolysis. Thus, the oils or fats ethanolysis reactions are basically carried out to obtain an important reduction in the viscosity of these materials, as they share similar values in all other chemical–physical significant parameters with the fossil diesel, except the viscosity, which was in the range 28–40 mm2/s or cSt values, while fossil diesel exhibits in the range 2–6 cSt values. Kinematic viscosity was determined in a capillary viscometer Oswald Proton Cannon-Fenske Routine Viscometer 33200, size 150. This is based on determining the time needed for a given volume of fluid passing between two points marked on the instrument. The kinematic viscosity (y, centistokes, cSt) can be obtained from the equation: C t = y, where C is the constant calibration of the measuring system in cSt s, which is given by the manufacturer (0.04058 mm2 s 1, at 408C) and t the flow time in seconds [10–14]. The sample is previously centrifuged at 3500 rpm during 10 min and filtered before its introduction in the viscometer. Then this is introduced in the water bath at 408C during 10 min before viscosity measurement [10–14].
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enzymatic activity, r = 1.93 mmol/min gBiocat, or what is the same, the processing of 2.3 mL of sunflower oil per min per g of Lypozyme.
Influence of the water content
Research Paper FIGURE 3
Influence of the amount of Lipozyme RM IM lipase on the conversion in the selective enzymatic ethanolysis of sunflower oil, operating under standard reaction conditions.
50 ml of aqueous solution of 10 N NaOH. The results are shown in Fig. 3. Here we can see a maximum for the conversion it is obtained for the interval 40–45 mg of the Lypozyme amount, which corresponds to 0.5 wt% with respect to the oil used in the reactions. Thus, a linear increase in the reaction conversion values with increasing the Lipozyme amount to a maximum value with 40 mg is obtained. From here, a slow decrease in conversion is obtained when the amount of the biocatalyst is increased. This decrease has no clear kinetic explanation, as a practically constant value would be expected, or even a slight increase in the conversions with the increase of the biocatalyst weight over the maximum obtained. This would indicate that, under the experimental conditions employed, the limit where the kinetic control of the enzymatic process operates is that obtained with 40 mg of biocatalyst weight. Probably, this decrease could be caused by the formation of aggregates by the polymer beads where lipases are immobilized. Larger particles of immobilized biocatalyst would hinder the mass transfer lowering the conversion. In summary, the experimental conditions optimized are 40 8C and 0.5 wt% of Lypozyme respect to sunflower oil, lets obtain a maximum reaction rate for the Lipozyme biocatalyst or specific
Currently waste cooking oil is a very important raw material for biofuel production but they have an important drawback associated to the water content. This parameter may affect the enzymatic process due to the very variable values of water activity exhibited by the various lipases from different sources [10,11,25]. Thus, this parameter needs to be determined to be able to evaluate the aptitude of Lipozyme to operate with waste oils, which usually contain variable amounts of water. To evaluate the influence of the water activity in the biocatalyst, a series of experiments are carried out operating under the optimal experimental conditions (temperature of 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, oil/ethanol volume ratio 12/3.5 (mL) and 40 mg of immobilized R. miehei lipase) and introducing in each experiment increasing deionized water amounts. Results obtained are shown in Table 1. It can be seen that the added water amount to the reaction promotes a lowering in conversion values. As can be expected, in Fig. 4 the parallel increase is seen in the kinematic viscosity with the water amount present in the reaction medium. This behavior may constitute a considerable handicap for its application in the enzymatic production of biofuels using waste cooking oils that generally contain water amounts higher than 0.2 wt%. In this respect it would imply a previous dehydration treatment before its use as raw material in the ethanolysis process.
Reuse of Lipozyme RM IM To study the biocatalytic behavior of Lipozyme RM IM along its successive reuse in the selective transesterification reaction of sunflower oil with absolute ethanol, a series of experiments with successive reactions were carried out under optimized standard conditions: oil/ethanol volume ratio 12/3.5 (mL), at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, reaction temperature 408C, 40 mg of Lipozyme RM IM. Before every reuse of the commercial immobilized lipase, this is subjected to washing treatment with a 100 mM phosphate buffer solution at pH 8, and dried in a desiccator for 2 hours. After washing and drying of the immobilized lipase, it is reused in a
TABLE 1
Influence of the water content in the enzymatic ethanolysis reaction of sunflower oil carried out with Lipozyme RM IM lipase, under the optimized conditions: oil/ethanol 12/3.5 volume ratio, 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH and 40 mg of immobilized lipase Kinematic viscosity (mm2/s)
Water content (%)
Conversion (%)
FAEE + MG (%)
DG (%)
TG (%)
0.0
78.5
59.8
18.7
21.5
8.3
0.05
75.3
60.1
15.2
24.7
8.6
0.10
76.8
58.7
18.1
23.2
8.8
0.16
77.4
58.1
19.3
22.6
9.7
0.22
61.7
49.2
12.5
38.3
10.5
0.27
38.7
30.1
8.3
61.6
14.2
4
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FIGURE 4
FIGURE 5
Influence of the amount of water amount on the conversion and viscosity in the selective enzymatic ethanolysis of sunflower oil with Lipozyme RM IM lipase, operating under standard reaction conditions.
Residual activity and viscosity obtained after reusing the enzyme in the selective enzymatic ethanolysis of sunflower oil, operating under standard reaction conditions.
TABLE 2
Influence of the biocatalyst reuse in the enzymatic ethanolysis reaction of sunflower oil carried out with Lipozyme RM IM lipase, under the optimized conditions: relative oil/ethanol 12/3.5, 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH and 40 mg of immobilized lipase, after washing treatment with a 100 mM phosphate buffer solution at pH 8, and dried in a desiccator for 2 hours Use N8
Kinematic viscosity (mm2/s)
Conversion (%)
FAEE + MG (%)
DG (%)
TG (%)
1
88.6
61.7
26.9
11.4
8.3
2
74.6
60.3
14.4
25.4
8.6
3
81.1
59.5
21.6
18.9
8.7
4
80.5
60.9
19.6
19.5
8.7
5
67.4
53.1
14.3
32.6
8.9
6
56.1
46.8
9.3
43.9
9.4
7
48.4
38.2
10.1
51.6
11.4
8
45.1
33.8
11.3
54.9
14.1
9
38.3
29.6
8.7
61.7
14.5
10
36.6
28.2
8.4
63.4
16.1
new reaction. The reaction products are chromatographically analyzed and the viscosity values determined. All these data are collected in Table 2. In this respect, it can be seen in Fig. 5 that the slight initial decrease in conversion values seems to be stabilized until the fourth use, to subsequently follow almost linearly descending to the seventh use. After the seventh use appears a tendency to stabilizing at very low conversion values, about the order of 20%, which seems kept as residual activity. It can also be seen in Fig. 5 that the kinematic viscosity undergoes an exponential increase along the reuses, thus limiting the reuse of immobilized R. miehei lipase practically beyond six consecutive reactions. The results obtained with the current immobilized lipases exhibits comparable conversion values (80–90%) to those described with covalently immobilized PPL [12–14]; however,
there are important differences in the behavior with respect to the reuses. Thus, the immobilized commercial enzyme activity decreased very quickly compared with the PPL covalently immobilized. However, the Lipozyme RM 1 M behavior is comparable with those of PPL immobilized by physical retention on demineralized sepiolite [10]. This similar behavior may be explained because in both cases lipases are retained by forces of physical character.
Conclusions To improve a new methodology to keep glycerol as MG, 1,3 selective biocatalyst Lipozyme RM IM has been identified and evaluated as useful, an immobilized R. miehei lipase in beads from macroporous anion exchange resins. Thus, it has been confirmed that this immobilized lipases allow its application to the production of biodiesel-like biofuels that keeps glycerol as
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monoglycerides, by using the selective character of the 1.3 selective R. miehei lipases immobilized in beads of interchange anion resins. In this respect, in no case the presence of glycerol was obtained as a reaction product, whatever the obtained yields, in the different studied enzymatic processes. We have studied the conditions of operation and most influential variables in the biocatalyzed selective ethanolysis reaction, allowing five or six reuses of the biocatalyst with acceptable yields. This new biofuel enzymatically obtained can be blended in any proportion with Diesel and be used directly in any diesel engine. Accordingly, the 1,3-selective behavior of these lipases allows a new methodology
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to obtain an alternative biodiesel using an enzymatic approach that is technically feasible and economically viable.
Acknowledgements Grants from the Spanish Ministry of Economy and Competitiveness (Project ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ201128954-C02-02), FEDER funds and Junta de Andalucı´a FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully acknowledged by the authors. We are also grateful to Novozymes AS, Denmark, for kindly supplying Lipozyme RM IM lipase.
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Selective ethanolysis of sunflower oil with Lipozyme RM IM, an immobilized Rhizomucor miehei lipase, to obtain a biodiesel-like biofuel, which avoids glycerol production through the monoglyceride formation Juan Calero1, Cristo´bal Verdugo2, Diego Luna1, Enrique D. Sancho3, Carlos Luna1, Alejandro Posadillo4, Felipa M. Bautista1 and Antonio A. Romero2 1
Department of Organic Chemistry, University of Cordoba, Campus de Rabanales, Ed. Marie Curie, 14014 Co´rdoba, Spain Crystallographic Studies Laboratory, Andalusian Institute of Earth Sciences, CSIC, Avd. Las Palmeras, n84, 18100 Armilla, Granada, Spain 3 Department of Microbiology, University of Co´rdoba, Campus de Rabanales, Ed. Severo Ochoa, 14014 Co´rdoba, Spain 4 Seneca Green Catalyst S.A., Edif. Centauro, Parque Cientı´fico Tecnolo´gico de Co´rdoba, Rabanales 21, 14014 Co´rdoba, Spain 2
The obtaining of Ecodiesel, a biofuel applicable to diesel engines which keeps the glycerin as monoglyceride (MG), was achieved through a selective ethanolysis process of sunflower oil, by application of Lipozyme RM IM, a Rhizomucor miehei lipase immobilized on macroporous anion exchange resins. This biocatalyst that was already described in the synthesis of conventional biodiesel has also shown its efficiency in the present selective enzymatic process, after optimization of the influence of various reaction parameters. Thus, an adequate activity is obtained that is maintained throughout five successive reuses. Quantitative conversions of triglycerides (TG) with high yields to fatty acid ethyl esters (FAEE) were obtained under mild reaction conditions that correspond to the transformation of TG in a mixture of two moles of FAEE and a mole of MG, thus avoiding the glycerol production. Thus, the selective transesterification reaction of sunflower oil with absolute ethanol can be carried out under standard conditions with oil/ethanol volume ratio 12/3.5 (mL), at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, reaction temperature of 408C and 40 mg of Lipozyme RM IM. Under these experimental conditions six successive reactions can be efficiently carried out.
Introduction Nowadays the fossil fuel is the main primary source of energy. However, the worldwide demand for fuel is increasing (about 90 million barrels per day) and fossil fuel availability is becoming more limited; hence, it is becoming more accepted that the cheap and easily accessible fossil fuel era is coming to its end [1]. Thus, in the medium term obtaining alternative energies that are environmentally and economically viable [2] are essential. In the past years the biodiesel has been in this respect considerate as the most appropriate alternative to the fossil fuel. Thus, several methods have been reported for the biodiesel production from vegetable or waste cooking oils and animal fats [3]. In this respect, the vegetable Corresponding author:. Calero, J. ([email protected], [email protected])
oil has all the chemical–physical parameters similar to the fossil fuel; however, the viscosity is higher than petroleum diesel fuel in the order of 10–20 times. Thus, direct use of vegetable oil is not applicable to most of the actual diesel engines [4]. To reduce the viscosity of vegetable oil the methodology most accepted is the transesterification reaction for biodiesel production [5]. To obtain biodiesel an excess of methanol is normally used to shift the equilibrium to the production of fatty acid methyl esters (FAMEs). Along the three steps process, glycerol is obtained as a by-product. However, this glycerol is the main disadvantage of this method, not only because of reduction in the atomic yield of the process (approximately 10 per cent), but also the biodiesel obtained needs to be cleaned of the waste glycerol. In this cleaning process a lot of water is used [6].
www.elsevier.com/locate/nbt 1 Please cite this article in press as: Calero, J. et al., Selective ethanolysis of sunflower oil with Lipozyme RM IM, an immobilized Rhizomucor miehei lipase, to obtain a biodiesel-like biofuel, which avoids glycerol production through the monoglyceride formation, New Biotechnol. (2014), http://dx.doi.org/10.1016/j.nbt.2014.02.008
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To avoid the associated problems with the generation of glycerine, several alternative methods are in research to obtain glycerol derivatives, in the same transesterification reaction. These methodologies avoid the separation of glycerine, by incorporating some glycerol derivative in the reaction products [7]. Thus, mixtures of FAMEs and glycerol triacetate (triacetin) are products of the interesterification reaction of triglycerides with methyl acetate in the presence of strong acid catalysts, so that all these products could be used as components of new type of a patented novel biofuel, which strongly improves economy of the biofuel production. It is claimed that such a mixture named Gliperol exhibits fuel characteristics comparable to the traditional biodiesel [8]. The reaction between triglycerides and DMC produces a mixture of FAMEs and cyclic esters glycerol carbonate (GC) of fatty acid, FAGCs, which constitutes a novel biodiesel-like material, named DMC-BioD [9]. The interesterification reaction of triglycerides with dimethyl carbonate (DMC) can generate a mixture of FAMEs, FAGCs molecules and GC. By contrast, a protocol for the preparation of a new biodiesel that keeps glycerol into their composition as monoacylglycerols (MG) is developed via 1,3-regiospecific enzymatic transesterification of sunflower oil using lipases, free [10,11] and immobilized [12–14]. Thus, the already patented Ecodiesel is obtained through the 1,3-selective partial ethanolysis of the triglycerides with PPL (that stops the process in the second step), according to Fig. 3. In this way, a mixture of two parts of FAEE and one part of MG is obtained. This is a soluble product in the diesel fuel and exhibits furthermore better lubricity than conventional biodiesel [15–17]. However, the Ecodiesel term is currently ascribed to whichever blend of fatty acid alkyl ester with ethanol, alone or with any proportion of diesel fuel [18–21]. This biofuel named Ecodiesel is able to directly operate diesel engines, alone or in whichever mixture with diesel fuel, without any separation or purification process (Fig. 1). To improve the viability and competitiveness of the enzymatic process to obtain Ecodiesel, the present study aims to evaluate in this process the commercial biocatalyst Lipozyme RM IM, a Rhizomucor miehei lipase immobilized in macroporous anion exchange resins, which has already been described in the synthesis of conventional biodiesel [22] as well as in biodiesel-like biofuels by interesterification with DMC [23,24] or methyl acetate [24]. Thus, in this study we attempt to put in value the 1,3 selective behavior of this commercial immobilized R. miehei lipase as well as
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the possibility of its reuse, to make profitable the production of alternative biofuels that avoids the glycerol generation, using an enzymatic approach. In this regard, this study is conducted to evaluate the influence of various reaction parameters on the activity of this immobilized R. miehei lipase as well as the reuse possibilities, to overcome the existing limitations in the use of industrial lipases, mainly associated with their high costs.
Materials and methods Materials Commercial sunflower oil was obtained locally. The ethyl esters of palmitic acid, stearic acid, oleic acid, linoleic acid and linolenic acid were from Accustandard, and the hexadecane (cetane) was obtained from Sigma–Aldrich and they all were chromatographically pure materials. Other chemicals like absolute ethanol and sodium hydroxide were obtained commercially from Panreac and they were pure analytical compounds (99%). The Lipozyme RM IM, a R. miehei lipase immobilized in beads from macroporous anion exchange resins, was kindly provided by Novozymes A/S.
Analytical method Reaction products were monitored by capillary column gas chromatography, using a Varian 430-GC gas chromatograph, connected to a HT5 capillary column (25 m 0.32 mm ID 0.1 mm, SGE, Supelco) with a flame ionization detector (FID) at 4508C and splitless injection at 3508C. As carrier gas helium is used, with a flow of 1.5 mL/min. A heating ramp from 908C to 2008C at a rate of 78C/min has been applied, followed by another ramp from 2008C to 3608C at a rate of 158C/min, maintaining the temperature of the oven at 3608C for 10 min. To determine the absence of glycerol as reaction product, the ramp was started from 458C. As internal standard n-hexadecane (cetane) is used to quantify the reaction conversion. The fatty acid ethyl esters (FAEE) and glycerides (monoglyceride, diglyceride and triglyceride – MG, DG and TG) are determined with the help of some commercial standard fatty acid esters. This method allows us to make a complete analysis of the sample in a single injection and in a time not higher than 60 min, which simplifies the process and increases the speed of analysis [10–14].
Kinematic viscosity measurements The viscosity is an important parameter for the correct running of diesel engines. In fact, the transesterification reaction is developed
FIGURE 1
New biodiesel-like biofuel (Ecodiesel) patented by University of Cordoba, obtained by lipase 1,3 selective ethanolysis of sunflower oil that integrated the glycerol as monoacylglycerols. It is formed by two moles of ethyl esters of fatty acids (FAEE) and one mole of monoglyceride (MG).
2
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Ethanolysis reactions To carry out the study the optimizations of operation conditions of Lipozyme RM IM, several parameters previously optimized with other lipases before being studied such as PPL and Lipopan 50 BG [10–14], have been maintained constant, to better study other variables more specifically dependent of every lipase, and that may produce important changes in the lipase activity, such as reaction temperature, lipase amount and mainly the water activity (aw). This latter one is very important to operate with waste oils, which usually contain variable amounts of water. Thus, a methodology OVAT (One Variable At Time) was used to study the influence of temperature, and it used enzyme loading and the water content in the reaction medium. These reactions were performed according to the experimental procedure previously described [10–14] to determine the optimal conditions for obtaining the selective ethanolysis reaction, such as amount of lipase, water amount and temperature operating at the same pH and oil/ethanol molar ratio (1/6). Thus, enzymatic assays are carried out with 9.4 g (12 mL, 0.01 mol) of commercial sunflower oil and 3.5 mL of absolute ethanol, at controlled temperatures (20–408C) in a 25 mL round bottom flask. Reaction mixtures were stirred with a conventional magnetic stirrer at a higher stirring speed than 300 rpm, to avoid mass transfer limitations, along a reaction time of 2 hours, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH. In this regard, a blank reaction in the presence of the highest quantity of solution of NaOH was performed to rule out a potential contribution from the homogeneous NaOH catalyzed reaction. Less than 10% conversion of the starting material was obtained, so that a homogenous base catalysis contribution can be considered as negligible under the investigated conditions. The different amounts of Lipozyme RM IM and commercial lipase are studied in the range of 0.05–0.1 g (0.1–1 wt%), temperatures between 30 and 708C and water
amounts from 0 to 0.3 wt%. Finally, a study of reuses is also carried out.
Results and discussion Influence of the reaction temperature To study the influence of the reaction temperature on the conversion values of the ethanolysis processes studied, a series of experiments are conducted with three different amounts of Lipozyme RM IM biocatalyst, in the range 20–100 mg, and at three different temperatures in the range 30–708C. Thus, these experiments are carried out under standard conditions, with an oil/ ethanol volume ratio 12/3.5 (mL) and constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH. The data obtained are shown in Fig. 2. From the results obtained a maximum activity at 408C for the three studied heterogeneous biocatalyst amounts can be easily seen. Similarly, a slight increase in the enzymatic activity can be seen when immobilized lipase amount increases from 20 to 50 mg, and then it falls drastically when 100 mg of Lipozyme is used. These results do not offer any doubt on the influence of temperature on the biocatalytic behavior of Lipozyme, clearly showing that the optimum operating temperature is 408C. However a more detailed assessment of the influence of the content of lipase is needed to determine the optimal biocatalyst amount to obtain maximum conversion per gram biocatalyst, that is, the specific enzymatic activity of Lipozyme in the selective ethanolysis process is studied.
Influence of the lipase amount To obtain more accurate information on the optimal value to optimize the conversion, the influence of the biocatalyst amount in the reaction yield is evaluated by increasing the lipase Lipozyme RM IM amount from 0.1 to 1 wt%, with respect to the oil weight and at the optimum value of temperature obtained. Thus, these experiments are carried out at 408C, with an oil/ethanol volume ratio 12/3.5 (mL) and constant pH obtained by the addition of
FIGURE 2
Influence of the reaction temperature on the conversion in the selective enzymatic ethanolysis of sunflower oil, using different amounts of Lipozyme RM IM lipase, operating under standard reaction conditions.
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Research Paper
to obtain a noticeable reducing in the viscosity, thus this biofuel can be used in the diesel engines. Thus, accurate viscosity measurements are crucial to assess the quality of biofuels produced; therefore, the characterization of this parameter is essential to evaluate the result obtained in the process of ethanolysis. Thus, the oils or fats ethanolysis reactions are basically carried out to obtain an important reduction in the viscosity of these materials, as they share similar values in all other chemical–physical significant parameters with the fossil diesel, except the viscosity, which was in the range 28–40 mm2/s or cSt values, while fossil diesel exhibits in the range 2–6 cSt values. Kinematic viscosity was determined in a capillary viscometer Oswald Proton Cannon-Fenske Routine Viscometer 33200, size 150. This is based on determining the time needed for a given volume of fluid passing between two points marked on the instrument. The kinematic viscosity (y, centistokes, cSt) can be obtained from the equation: C t = y, where C is the constant calibration of the measuring system in cSt s, which is given by the manufacturer (0.04058 mm2 s 1, at 408C) and t the flow time in seconds [10–14]. The sample is previously centrifuged at 3500 rpm during 10 min and filtered before its introduction in the viscometer. Then this is introduced in the water bath at 408C during 10 min before viscosity measurement [10–14].
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enzymatic activity, r = 1.93 mmol/min gBiocat, or what is the same, the processing of 2.3 mL of sunflower oil per min per g of Lypozyme.
Influence of the water content
Research Paper FIGURE 3
Influence of the amount of Lipozyme RM IM lipase on the conversion in the selective enzymatic ethanolysis of sunflower oil, operating under standard reaction conditions.
50 ml of aqueous solution of 10 N NaOH. The results are shown in Fig. 3. Here we can see a maximum for the conversion it is obtained for the interval 40–45 mg of the Lypozyme amount, which corresponds to 0.5 wt% with respect to the oil used in the reactions. Thus, a linear increase in the reaction conversion values with increasing the Lipozyme amount to a maximum value with 40 mg is obtained. From here, a slow decrease in conversion is obtained when the amount of the biocatalyst is increased. This decrease has no clear kinetic explanation, as a practically constant value would be expected, or even a slight increase in the conversions with the increase of the biocatalyst weight over the maximum obtained. This would indicate that, under the experimental conditions employed, the limit where the kinetic control of the enzymatic process operates is that obtained with 40 mg of biocatalyst weight. Probably, this decrease could be caused by the formation of aggregates by the polymer beads where lipases are immobilized. Larger particles of immobilized biocatalyst would hinder the mass transfer lowering the conversion. In summary, the experimental conditions optimized are 40 8C and 0.5 wt% of Lypozyme respect to sunflower oil, lets obtain a maximum reaction rate for the Lipozyme biocatalyst or specific
Currently waste cooking oil is a very important raw material for biofuel production but they have an important drawback associated to the water content. This parameter may affect the enzymatic process due to the very variable values of water activity exhibited by the various lipases from different sources [10,11,25]. Thus, this parameter needs to be determined to be able to evaluate the aptitude of Lipozyme to operate with waste oils, which usually contain variable amounts of water. To evaluate the influence of the water activity in the biocatalyst, a series of experiments are carried out operating under the optimal experimental conditions (temperature of 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, oil/ethanol volume ratio 12/3.5 (mL) and 40 mg of immobilized R. miehei lipase) and introducing in each experiment increasing deionized water amounts. Results obtained are shown in Table 1. It can be seen that the added water amount to the reaction promotes a lowering in conversion values. As can be expected, in Fig. 4 the parallel increase is seen in the kinematic viscosity with the water amount present in the reaction medium. This behavior may constitute a considerable handicap for its application in the enzymatic production of biofuels using waste cooking oils that generally contain water amounts higher than 0.2 wt%. In this respect it would imply a previous dehydration treatment before its use as raw material in the ethanolysis process.
Reuse of Lipozyme RM IM To study the biocatalytic behavior of Lipozyme RM IM along its successive reuse in the selective transesterification reaction of sunflower oil with absolute ethanol, a series of experiments with successive reactions were carried out under optimized standard conditions: oil/ethanol volume ratio 12/3.5 (mL), at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH, reaction temperature 408C, 40 mg of Lipozyme RM IM. Before every reuse of the commercial immobilized lipase, this is subjected to washing treatment with a 100 mM phosphate buffer solution at pH 8, and dried in a desiccator for 2 hours. After washing and drying of the immobilized lipase, it is reused in a
TABLE 1
Influence of the water content in the enzymatic ethanolysis reaction of sunflower oil carried out with Lipozyme RM IM lipase, under the optimized conditions: oil/ethanol 12/3.5 volume ratio, 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH and 40 mg of immobilized lipase Kinematic viscosity (mm2/s)
Water content (%)
Conversion (%)
FAEE + MG (%)
DG (%)
TG (%)
0.0
78.5
59.8
18.7
21.5
8.3
0.05
75.3
60.1
15.2
24.7
8.6
0.10
76.8
58.7
18.1
23.2
8.8
0.16
77.4
58.1
19.3
22.6
9.7
0.22
61.7
49.2
12.5
38.3
10.5
0.27
38.7
30.1
8.3
61.6
14.2
4
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FIGURE 4
FIGURE 5
Influence of the amount of water amount on the conversion and viscosity in the selective enzymatic ethanolysis of sunflower oil with Lipozyme RM IM lipase, operating under standard reaction conditions.
Residual activity and viscosity obtained after reusing the enzyme in the selective enzymatic ethanolysis of sunflower oil, operating under standard reaction conditions.
TABLE 2
Influence of the biocatalyst reuse in the enzymatic ethanolysis reaction of sunflower oil carried out with Lipozyme RM IM lipase, under the optimized conditions: relative oil/ethanol 12/3.5, 408C, at constant pH obtained by the addition of 50 ml of aqueous solution of 10 N NaOH and 40 mg of immobilized lipase, after washing treatment with a 100 mM phosphate buffer solution at pH 8, and dried in a desiccator for 2 hours Use N8
Kinematic viscosity (mm2/s)
Conversion (%)
FAEE + MG (%)
DG (%)
TG (%)
1
88.6
61.7
26.9
11.4
8.3
2
74.6
60.3
14.4
25.4
8.6
3
81.1
59.5
21.6
18.9
8.7
4
80.5
60.9
19.6
19.5
8.7
5
67.4
53.1
14.3
32.6
8.9
6
56.1
46.8
9.3
43.9
9.4
7
48.4
38.2
10.1
51.6
11.4
8
45.1
33.8
11.3
54.9
14.1
9
38.3
29.6
8.7
61.7
14.5
10
36.6
28.2
8.4
63.4
16.1
new reaction. The reaction products are chromatographically analyzed and the viscosity values determined. All these data are collected in Table 2. In this respect, it can be seen in Fig. 5 that the slight initial decrease in conversion values seems to be stabilized until the fourth use, to subsequently follow almost linearly descending to the seventh use. After the seventh use appears a tendency to stabilizing at very low conversion values, about the order of 20%, which seems kept as residual activity. It can also be seen in Fig. 5 that the kinematic viscosity undergoes an exponential increase along the reuses, thus limiting the reuse of immobilized R. miehei lipase practically beyond six consecutive reactions. The results obtained with the current immobilized lipases exhibits comparable conversion values (80–90%) to those described with covalently immobilized PPL [12–14]; however,
there are important differences in the behavior with respect to the reuses. Thus, the immobilized commercial enzyme activity decreased very quickly compared with the PPL covalently immobilized. However, the Lipozyme RM 1 M behavior is comparable with those of PPL immobilized by physical retention on demineralized sepiolite [10]. This similar behavior may be explained because in both cases lipases are retained by forces of physical character.
Conclusions To improve a new methodology to keep glycerol as MG, 1,3 selective biocatalyst Lipozyme RM IM has been identified and evaluated as useful, an immobilized R. miehei lipase in beads from macroporous anion exchange resins. Thus, it has been confirmed that this immobilized lipases allow its application to the production of biodiesel-like biofuels that keeps glycerol as
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monoglycerides, by using the selective character of the 1.3 selective R. miehei lipases immobilized in beads of interchange anion resins. In this respect, in no case the presence of glycerol was obtained as a reaction product, whatever the obtained yields, in the different studied enzymatic processes. We have studied the conditions of operation and most influential variables in the biocatalyzed selective ethanolysis reaction, allowing five or six reuses of the biocatalyst with acceptable yields. This new biofuel enzymatically obtained can be blended in any proportion with Diesel and be used directly in any diesel engine. Accordingly, the 1,3-selective behavior of these lipases allows a new methodology
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to obtain an alternative biodiesel using an enzymatic approach that is technically feasible and economically viable.
Acknowledgements Grants from the Spanish Ministry of Economy and Competitiveness (Project ENE 2011-27017), Spanish Ministry of Education and Science (Projects CTQ2010-18126 and CTQ201128954-C02-02), FEDER funds and Junta de Andalucı´a FQM 0191, PO8-RMN-03515 and P11-TEP-7723 are gratefully acknowledged by the authors. We are also grateful to Novozymes AS, Denmark, for kindly supplying Lipozyme RM IM lipase.
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