Biodiesel Synthesis Catalyzed by Burkholderia cenocepacia Lipase

Feb 4, 2011 - Biodiesel Synthesis Catalyzed by Burkholderia cenocepacia Lipase Supported on Macroporous Resin NKA in Solvent-Free and Isooctane System...
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Biodiesel Synthesis Catalyzed by Burkholderia cenocepacia Lipase Supported on Macroporous Resin NKA in Solvent-Free and Isooctane Systems Yun Liu,* Tao Liu,† Xiaofeng Wang, Li Xu, and Yunjun Yan* Key Laboratory of Molecular Biophysics of the Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, People's Republic of China ABSTRACT: Enzymatic biodiesel production was investigated in solvent-free and isooctane systems using Burkholderia cenocepacia lipase (BCL) immobilized on macroporous resin NKA as a biocatalyst. A total of 98% biodiesel yield was obtained under the optimized conditions of methanol/oil molar ratio 4:1 with the addition of methanol in three steps at intervals of 2 h, enzyme dosage 2.5 wt % (based on the oil weight), moisture concentration 7 wt % (based on oil weight), reaction temperature 40 °C, reaction duration 8 h, stirring rate 300 rpm, and isooctane amount 50 wt % (based on the oil weight). Compared to tert-butanol and solventfree systems, the highest biodiesel yield was achieved in the isooctane system. BCL-NKA showed high operational stability with no obvious loss in lipase activity for transesterification in the isooctane system even after 50 cycles (400 h) of repeated usage. It has been revealed that BCL-NKA-catalyzed transesterification in the isooctane system is a promising alternative for biodiesel production.

1. INTRODUCTION Because of its nontoxic, environmentally friendly, and mild operating conditions, enzymatic catalysis for biodiesel production has recently become the more attractive option compared to the chemical catalysis method.1 Data on lipases employed for biodiesel production were easily available in the literature, such as commercial Novozym 435,2 Lipozyme TLIM,3 lipase PS,4 Lipozyme RMIM,5 etc. However, particular attention has been paid to lipase PS, the lipase from Burkholderia cepacia because of its high stability, alcohol tolerance, and activity suitable for a broad spectrum of reactions, substrates, and media.6 Burkholderia cenocepacia, a genomovar level of B. cepacia, was directly screened by a TB-TA plate and identified by HaeIII-recA RFLP (restriction fragment length polymorphism) and genomovar-specific PCR in our laboratory.7 Experiments revealed that the lipase from B. cenocepacia (BCL) is a versatile lipase for biodiesel production. It has been demonstrated that more than 1/2 mol equiv of methanol is insoluble in vegetable oils and the lipases are easily inactivated by contact with methanol existing as drops in the oils. Furthermore, the byproduct glycerol in biodiesel production is also easily adsorbed onto the surface of the lipases, which also leads to a negative effect on the lipase activity and operational stability. Because the lipase might be inactivated by the insoluble methanol and glycerol in the system, it is reasonable to solve the problem through solvent selection to improve methanol and glycerol solubility.8 tert-Butanol is a good solvent of the substrate methanol, and many previous studies have been focused on this solvent.9-11 Apart from tert-butanol, many other solvents such as n-hexane,12 ionic liquids,13 tert-amyl alcohol,14 isooctane,15 and cosolvent mixtures16 were all employed as the reaction systems for lipasecatalyzed alcoholysis for biodiesel production. Therefore, research on isooctane as the solvent for lipase-catalyzed transesterification for biodiesel production can be available in the literature.17 The authors reported that pretreatment of the membrane-bound lipase with r 2011 American Chemical Society

isooctane could be an effective method for enhancing its synthesis activity, such as lipase from Rhizopus chinensis. As a versatile lipase for biodiesel production, there is no report on the effect of isooctane on the conversion activity of BCL available so far. On the basis of the above-mentioned analysis, we have chosen isooctane as the solvent to investigate its effect on the biodiesel synthesis by a BCL catalyst in this work. Immobilization of enzymes inside the porous structure of a solid (resin NKA in our work) may permit one to have the enzyme molecules fully dispersed without the possibility of interacting with any external interface. Thus, this immobilization will stabilize the enzyme against interaction with molecules from the enzymatic extract, preventing aggregation, autolysis, or proteolysis by proteases from the extract (which will also be dispersed and immobilized).18 Moreover, we have investigated 11 types of macroporous resins upon lipase activity recovery, such as NKA, AB-8, HP, XAD, D699K, D380, D418, D311, D113, D401, and D152. The results show that the lipase activity recovery (125%) is highest when lipase is immobilized on macroporous resin NKA. On the other hand, resin NKA is inert. It shows no catalytic activity for transesterification/esterification reactions. Therefore, resin NKA has been chosen as the immobilization matrix in our work. According to the above-mentioned analysis, we aim to use BCL immobilized on macroporous resin NKA (BCL-NKA) as the biocatalyst for methanolysis of soybean oil for biodiesel production in solvent-free and isooctane systems. The main objectives of this work are to investigate the effect of the crucial variables on the biodiesel yield, such as BCL dosage [1.5-4.5 wt % (based on the oil weight, g)], molar ratio of alcohol/oil (from 3:1 to 8:1), moisture content [up to 10 wt % (based on the oil weight, g)], reaction temperature (from 30 to 55 °C), reaction duration (up to 12 h), amount of reaction medium [up to 80 wt % (based on the Received: October 22, 2010 Published: February 04, 2011 1206

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Energy & Fuels oil weight, g)], and methanol addition approach [(1) methanol is added in one step; (2) methanol is added in two steps at an interval of 4 h; (3) methanol is added in three steps at intervals of 2 h]. We also compare the effect of three reaction media on the biodiesel yield catalyzed by BCL-NKA, such as solvent-free, tert-butanol, and isooctane systems under the same operational conditions. Furthermore, the operational stability of BCL-NKA in the isooctane system is finally addressed. In this work, soybean oil is chosen as the feedstock for the following main reasons: (i) It is a major domestic crop in China. Soybean oil is one of the four vegetable oils, e.g., rapeseed oil, soybean oil, peanut oil, and corn oil. The total soybean production in China in 2005 was 17.4 million ton (FAO, 2005), which was ranked fifth in the world. (ii) Numerous reports on transesterification of soybean oil to produce biodiesel are available in the literature. So, we can easily compare the catalytic activity of BCL-NKA with other enzyme catalysts under similar conditions.

2. EXPERIMENTAL SECTION Materials. B. cenocepacia, a genomovar level of B. cepacia, was directly screened by a TB-TA plate and identified by HaeIII-recA RFLP (restriction fragment length polymorphism) and genomovar-specific PCR in our laboratory.7 Macroporous resin NKA with a particle size of 0.3-1.25 mm was bought from Tianjin Nankai Science & Technology Co. Ltd. (Tianjing, China). Methanol, tert-butanol, and isooctane were of analytical grade and from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Triton X-100 and sodium dodecyl sulfate (SDS) were bought from Tianjin Chemical Reagent Co. Ltd. (Tianjin, China). Reference standards of fatty acid methyl esters (FAMEs), including methyl tridecanoate, methyl laurate, methyl palmitate, methyl heptadecanoate, methyl stearate, methyl oleate, methyl linoleate, and methyl linolenate, were from the Sigma Chemical Co. Ltd. (St. Louis, MO). BCL Immobilization on Macroporous Resin NKA. The procedures of BCL immobilization on macroporous resin matrix NKA are described as follows: 1 g of resin was immersed in 5 mL of absolute ethanol in a 25 mL test tube for 4 h. After evaporation of ethanol, it was eluted six times with distilled water and placed in a beaker filled with 5 mL of 0.05 M phosphate buffer (pH 7) overnight at 4 °C, and then the buffer was filtered and the resin was retained in the tube. After resin pretreatment, 5 mL of 0.05 M phosphate buffer (pH 7) containing 0.8 g of free BCL powder was added to the tube and the mixture was stirred in a rotary shaker with a stirring speed of 200 rpm at 37 °C. After 2 h, the suspension was filtered through a Buchner funnel. BCL adsorbed on the resin matrix was washed five times with 5 mL of 0.05 M phosphate buffer (pH 7) to remove unadsorbed enzyme, and then it was dried in a FD-1D-50 vacuum desiccator. To further confirm the fastness between BCL and resin NKA, 20% Triton X-100 and SDS were employed to treat the BCL-NKA catalyst using ultrasonic assistance for 3 h.19 While the BCL-NKA catalyst was treated using 20% Triton X-100 and SDS, after filtering, the protein concentration of the supernatant was measured to provide direct evidence of robustness between BCL with the resin NKA matrix. Methanolysis of Soybean Oil for Biodiesel Production by BCL-NKA in the Isooctane System. Transesterification reactions by BCL-NKA were conducted in a 50 mL shaking flask in a thermostat shaking bed with a stirring rate of 200 rpm. The reaction mixture consisted of soybean oil, BCL-NKA lipase, methanol, and isooctane. The influence of crucial operational parameters such as BCL-NKA dosage [1.5-4.5 wt % (based on the oil weight, g)], molar ratio of alcohol/oil (from 3:1 to 8:1), moisture content [up to 10 wt % (based on the oil weight, g)], reaction temperature (from 30 to 55 °C), reaction duration (up to 12 h), amount of reaction medium [up to 80 wt % (based

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on the oil weight, g)], and methanol addition approach [(1) methanol is added in one step; (2) methanol is added in two steps at an interval of 4 h; (3) methanol is added in three steps at intervals of 2 h] on the biodiesel yield was systematically studied. Enzymatic transesterification reactions in the solvent-free system under the same conditions were employed as the controls. After a certain reaction period, 50 μL samples were taken from the reaction mixture and centrifuged. Then 10 μL withdrawn from the supernatant and 290 μL of 1.0 mM methyl heptadecanoate (as the internal standard) were precisely measured and mixed thoroughly for gas chromatography (GC) analysis for the biodiesel yield. GC Determination of the Biodiesel Yield. The biodiesel yield was determined by a GC-9790 gas chromatograph (Fuli Analytical Instrument Co. Ltd., Wenlin, China) according to the method reported in our previous work.3 The column is an Agilent INNOWAX capillary column (30 m  0.25 mm i.d.  0.25 μm, J&W Scientific, Folsom, CA). The initial column temperature was 200 °C and kept for 2 min and then was raised to 235 °C at a rate of 3 °C/min and maintained for l min. The temperature of the injector and the flame ionization detector was 280 °C. The biodiesel yield was quantified in the presence of methyl heptadecanoate as the internal standard and other standard FAMEs. The biodiesel yield (%) is defined as the conversion of soybean oil and calculated as the total FAME content in the conversion oil sample with eqs 1 and 2 Asample f0 ð1Þ biodiesel yield ð%Þ ¼ Ainternal Winternal f0 ¼

Wsample Ainternal Winternal Asample

ð2Þ

where Asample is the peak area of free fatty acids in the sample, f0 is the response factor, Ainternal is the peak area of the internal standard, Winternal is the weight (g) of the internal standard, and Wsample is the weight (g) of the sample.

Determination of the Protein Concentration and Specific Activity of Lipase. Protein concentration determination of lipase was measured by the Bradford protein assay method using a Bradford reagent from biorad at a wavelength of 595 nm with a standard calibration curve of bovine serum albumin, which was detailed by Petkar et al.20 The specific activity of BCL was calculated according to the method in our previous work.16 One unit is the amount of enzyme that catalyzes the formation of 1 μmol of conversion in 1 min at 37 °C. Therefore, the specific activity (moles of conversion per gram per minute) was expressed as moles of ester formed per gram of protein per minute. Statistical Data. All reported data were collected in triplicate, and statistical analysis was performed using SAS 9.0 software (SAS Institute Inc., Cary, NC). Analytical data were expressed as average value ( standard deviation.

3. RESULTS AND DISCUSSION Influence of the Isooctane Addition Amount on the Biodiesel Yield. We investigated transesterification of soybean

oil with methanol catalyzed by BCL-NKA in the isooctane system. The results of the effect of the isooctane addition amount of up to 80 wt % (based on the oil weight) are shown in Figure 1A. As shown in Figure 1A, the highest biodiesel yield with 97.2% is observed at a isooctane concentration of 50 wt %. Therefore, the optimum isooctane addition amount is found to be 50 wt %, and the biodiesel yield gradually decreases beyond this concentration. This observation is contradictive with the results of the literature.15 The authors in the literature found that a low substrate conversion was obtained in a pure isooctane system for transesterification of Jatropha curcas L. seed oil with methanol by Novozym 1207

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Figure 1. Influence of the isooctane loading on the biodiesel yield [conditions: oil, 2 g; molar ratio of methanol/oil, 4:1; lipase loading, 2.5 wt % (based on the oil weight); reaction temperature, 40 °C; reaction duration, 8 h; stirring rate, 300 rpm; moisture content, 5 wt % (based on the oil weight)].

435. They speculated that the poor solubility of methanol in the isooctane solvent and the excessive insoluble methanol in the reaction medium gravely deactivates the lipase. In fact, we demonstrated that the hydrophobicity (log P), functional group, and molecular constitution of organic solvents are all crucial factors affecting the lipase activity. The catalytic activity variance of lipase from B. cepacia is significantly attributed to its conformational changes in organic solvents.21 On the basis of the above-mentioned analysis, a high biodiesel yield is obtained in the isooctane system with 50 wt % concentration; a reasonable explanation is ascribed to the fact that isooctane solvent molecules could interact with hydrophobic amino acid residues in the lid that covers the catalytic site of the enzyme, thereby maintaining enzyme in its open conformation and leading to high catalytic activity.22 However, because of the poor solubility of methanol and byproduct glycerol in the isooctane solvent, the deposit of methanol and glycerol coating BCL is formed with an further increase of the isooctane addition amount (more than 50 wt %), which will increase the mass-transfer and diffusion constraints as well as the inactivating lipase.23 This is the reason why the biodiesel yield gradually decreases when the isooctane addition amount is up to 80 wt %. Most studies deduced that only polar organic solvents, such as tert-butanol, have been used as proposed solvent engineering methods to solve the negative effects of methanol and glycerol on lipase.5,11,15 In this work, we compared the influences of three different solvent systems, such as the solvent-free, tert-butanol, and isooctane systems, on the activity of BCL-NKA. As shown in Figure 1B, the highest enzyme activity is observed in the isooctane system with ca. 93% biodisel yield, while the lowest enzyme activity is found in the tert-butanol system with only 67% biodiesel yield. A reasonable explanation is ascribed to the fact that tert-butanol strips the essential water off the lipase molecules and then inactivates the biocatalyst and thus influences the biodiesel yield during transesterification.24 It is suggested that, in choosing suitable reaction solvent systems, one should consider not only the hydrophobicity of the organic solvents but also the types of lipases, as well as the effect of the organic solvents on the conformation and microenvironment of lipase molecules. Influence of the BCL-NKA Dosage on the Biodiesel Yield. Experiments were carried out to investigate the effect of BCLNKA loading on the methanolysis of soybean oil for biodiesel production in solvent-free and isooctane systems (Figure 2). As shown in Figure 2, the biodiesel yield is enhanced by increasing the enzyme loading regardless of the solvent-free and isooctane systems. The highest biodiesel yield is 97.5% at 9 h with

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Figure 2. Influence of the BCL-NKA enzyme amount on the biodiesel yield [conditions: oil, 2 g; molar ratio of methanol/oil, 4:1; isooctane addition amount, 50 wt % (based on the oil weight); reaction temperature, 40 °C; reaction duration, 9 h; stirring rate, 300 rpm; moisture content, 5 wt % (based on the oil weight)].

Figure 3. Effect of the enzyme amount on the activity of immobilized enzyme: immobilizations were carried out at 160 mg of lipase/mL of PBS (pH 7.0) and 37 °C of water bath for 2 h.

2.5 wt % BCL-NKA loading in isooctane system, while 88.5% biodiesel yield is obtained at 9 h for 4.5 wt % BCL-NKA loading in the solvent-free system. It is indicated that BCL-NKA shows higher catalytic activity in the isooctane system than in the solvent-free system, which agrees well with the observation in section 3.1. To elucidate this phenomenon, the reaction rate is determined after normalizing it to the number of active sites present in the BCL-NKA catalyst. The initial reaction rate is 13 600 mol of conversion/mol of catalytic site per minute at 37 °C in the isooctane system, which is 40-fold of free BCL and 2-fold of that reported by Sakai et al.25 When the enzyme concentration and the mass of resin NKA are selected, the total amount of enzyme for lipase immobilization is dependent upon how much lipase powder is assigned to 1 g of resin NKA. In our work, the lipase concentration was 160 mg of native lipase)/mL of phosphatebuffered saline (PBS). The effect of the enzyme loading on the specific activity of BCL-NKA is shown in Figure 3. As shown in Figure 3, the maximum specific activity of lipase is approximately 3.5  105 μmol of conversion/g of protein/min at a loading of 8.0 mg of protein/g of BCL-NKA. On the other hand, the temporal dynamics of methanolysis of soybean oil by BCL-NKA in the isooctane system is shown in Figure 4. 1208

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Figure 4. Time course of the transesterification reaction [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); molar ratio of methanol/oil, 4:1; adding methanol in three steps at 2 h intervals; isooctane addition amount, 50 wt % (based on the oil weight); moisture concentration, 7 wt % (based on the oil weight); stirring rate, 300 rpm; reaction temperature, 40 °C].

Figure 5. Influence of the moisture concentration on the biodiesel yield [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); molar ratio of methanol/oil, 4:1; isooctane addition amount, 50 wt % (based on the oil weight); reaction temperature, 40 °C; stirring rate, 300 rpm; reaction duration, 8 h].

Time course curve shows that biodiesel formation of the temporal dynamics is linear within 0.5 h, from which the initial reaction rate could be calculated. Also, over 97% biodiesel yield is obtained at 9 h for methanolysis of soybean oil by BCL-NKA in the isooctane system. However, a reaction time longer than 9 h does not change the biodiesel yield (about 97%) any more because a relative equilibrium conversion is achieved. Influence of the Moisture Concentration on the Biodiesel Yield. The moisture concentration in the reaction mixture is a characteristic and one of the most important factors deciding the lipase-catalyzed transesterification reaction rate and yield of biodiesel synthesis. It has been reported that enzymes should require the addition of a small aliquot of water to retain their activities.26 Lipase shows the unique feature of acting as the interface between an aqueous phase and an organic phase, so the lipase activity generally depends on the interfacial area. With an increase of the water addition, the amount of water available for oil to form oil-water droplets increases; thus, this helps to maintain the lipase activity.27 The biodiesel yield as a function of the percentage moisture content (up to 10%) in solvent-free and isooctane systems was investigated, and the results are presented in Figure 5. As shown in Figure 5, it is indicated that the enzyme activity of BCL-NKA increases dramatically when 2% water is added to

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Figure 6. Influence of the reaction temperature on the biodiesel yield [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); molar ratio of methanol/oil, 4:1; isooctane addition amount, 50 wt % (based on the oil weight); moisture concentration, 7 wt % (based on the oil weight); stirring rate, 300 rpm; reaction duration, 8 h].

the reaction mixture, which supports the fact that a small aliquot of the water content is required to activate the enzyme. There is a similar tendency of the biodiesel yield as a function of the percentage moisture content (up to 10%) in the solvent-free and isooctane systems. The biodiesel yield increases with an increase of the water addition content, and the highest biodiesel yield is obtained at 7% water addition into the transesterification reaction, approximately 90% in the isooctane system and 86% in the solvent-free system. However, the moisture concentration in transesterification plays multiple roles, and it has positive and negative effects on the BCL-NKA catalytic activity. Adding excess water into the reaction mixture might make the lipase more flexible and lead to some unintended side reactions such as hydrolysis, especially in the transesterification process. Additionally, too much water facilitates enzyme aggregation, which leads to a decrease in the enzyme activity. Numerous studies demonstrated that the addition of a certain aliquot of moisture to the enzyme-catalyzed reaction mixture (this aliquot cannot shift the reaction equilibrium in favor of hydrolysis) increased the rate of fatty acid ester synthesis, and the addition of the moisture amount should depend on the feedstock oil, lipase type, immobilized support, and organic solvent employed.10,26,28 For example, commercial Novozym 435 lipase shows high catalytic activity for transesterification without extra water added into the reaction mixture.10 Influence of the Reaction Temperature on the Biodiesel Yield. The effect of the temperature in the range of 30-55 °C on methanolysis of soybean oil for biodiesel production by BCL-NKA is shown in Figure 6. Figure 6 shows that the optimum temperature for BCL-NKA is found to be 40 °C with biodiesel yields of approximately 96% and 93% in the isooctane and solvent-free systems, respectively. Regardless of the solvent-free and isooctane systems, it is observed that there was a rise in the biodiesel yield catalyzed by BCL-NKA (at a molar substrate ratio of 4:1) when the temperature was gradually increased from 30 to 40 °C, but further increments in temperature to 50 °C caused a decrease in the biodiesel yield. Numerial studies revealed that the optimum temperature for enzymatic transesterification results from the interplay between the operational stability of the biocatalyst and the rate of transesterification. It also depends on the alcohol/oil molar ratio, type of organic solvent, and thermostability of enzymatic preparation.3,22,26 Influence of the Molar Ratio of Methanol/Oil on the Biodiesel Yield. The molar excess of alcohol over fatty acids contained in triacylglycerols always increases the transesterification yield, but it 1209

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Figure 7. Influence of the molar ratio of oil/methanol on the biodiesel yield [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); isooctane addition amount, 50 wt % (based on the oil weight); moisture concentration, 7 wt % (based on the oil weight); reaction duration, 16 h; stirring rate, 300 rpm; reaction temperature, 40 °C].

can also inactivate the enzyme, in particular when the alcohol is insoluble in the reaction mixture (it forms an emulsion, and the size of the droplets depends on the intensity of stirring). High yields of biodiesel synthesis can be achieved at more than the stoichiometric molar ratio of alcohol/oil in the organic solvent system because the addition of an organic solvent to the mixture increases the solubility of alcohol, which protects enzymes from inactivation for the transesterification reaction.5,26 In our work, the methanol concentration is varied from 1:3 to 1:8 to investigate its effect on the biodiesel yield under the following conditions: oil, 2 g; lipase dosage, 2.5% (based on the oil weight); isooctane addition amount, 50%; moisture concentration, 7%; reaction duration, 8 h; reaction temperature, 40 °C; stirring rate, 300 rpm; adding methanol with one step. The results of the effect of the methanol/oil molar ratio on methanolysis of soybean oil by BCLNKA in solvent-free and isooctane systems are shown in Figure 7. As can be seen from Figure 7, the maximal biodiesel yield was obtained at a molar ratio of oil/methanol of 1:4, approximately 84% in the isooctane system and 77% in the solvent-free system. Usually, in an organic solvent system, a slight excess of alcohol (over the stoichiometric alcohol/oil molar ratio of 4-5:1) is necessary to achieve a satisfactory yield of the transesterification process.5,26 In our present work for one batch transesterification reaction, the effect of the alcohol/oil molar ratio on the biodiesel yield in solvent-free and isooctane systems shows no significant difference. However, taking into consideration the operational stability of BCL-NKA lipase for long running times, the importance of the organic solvent is obvious for enzymatic transesterification qfor biodiesel production, which will been adopted in the following experiments. Influence of the Methanol Addition Approach on the Biodiesel Yield. In order to eliminate the negative effect of methanol on the BCL-NKA lipase catalytic activity, experiments were conducted to investigate the influence of the methanol addition approach on the biodiesel yield. Methanol addition approaches are as follows: (1) methanol is added in one step; (2) methanol is added in two steps at an interval of 4 h; (3) methanol is added in three steps at intervals of 2 h. The effect of the methanol addition approach on methanolysis of soybean oil by BCL-NKA for biodiesel production in solvent-free and isooctane systems is shown in Figure 8. As can be seen in Figure 8, in the case of the solvent-free system, a high biodiesel yield is achieved when methanol is added in three steps at intervals of 2 h. This means that a small aliquot of methanol is added into the mixture at the beginning of the transesterification reaction, and then more than 98% biodiesel

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Figure 8. Effect of the methanol addition approach on the biodiesel yield [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); molar ratio of methanol/oil, 4:1; isooctane addition amount, 50 wt % (based on the oil weight); moisture concentration, 7 wt % (based on the oil weight); stirring rate, 300 rpm; reaction temperature, 40 °C]. 1 Step: methanol is added in one step. 2 Steps: methanol is added in two steps at an interval of 4 h. 3 Steps: methanol is added in three steps at intervals of 2 h.

Figure 9. Operational stability of BCL-NKA in the isooctane system [conditions: oil, 2 g; lipase loading, 2.5 wt % (based on the oil weight); molar ratio of methanol/oil, 4:1; adding methanol in three steps at 2 h intervals; isooctane addition amount, 50 wt % (based on the oil weight); moisture concentration, 7 wt % (based on the oil weight); reaction temperature, 40 °C; stirring rate, 300 rpm; reaction duration, 8 h].

yield is obtained after 10 h of reaction. With regard to the isooctane system, more than 97% biodiesel yield is also achieved after 8 h of reaction using the methanol addition approach in three steps at 2 h intervals. The minor difference between the results in solvent-free and isooctane systems is probably that the solubility of methanol in the reaction mixtures considerably increases when fatty acid esters appear with reaction processing.29 Moreover, the addition of the isooctane solvent to the reaction mixture increases the solubility of FAMEs; therefore, methanol can be used in higher concentrations with reaction processing, which protects BCL-NKA from inactivation in the one-batch methanolysis of soybean oil. Reusability of BCL-NKA in the Isooctane System. BCLNKA was reused directly without any treatment after 8 h of reaction in each cycle, the operational stability of the lipase in the isooctane system was investigated, and the results are shown in Figure 9. As can been seen from Figure 9, there is no obvious loss in the biodiesel yield even after BCL-NKA is reused for 50 cycles (400 h) in the isooctane system. However, BCL-NKA shows a 1210

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Energy & Fuels dramatic decrease in activity from 92% to 58% in the solvent-free system after 5 cycles under the same conditions as those in the isooctane system. A reasonable explanation is probably that excess methanol and much byproduct glycerol was adsorbed onto the surface of BCL-NKA because of the poor solubility of methanol and glycerol in feedstock oil, which leads to the quite short operational life of BCL-NKA. From a comparison of Figures 5-8, no significant differences of the biodiesel yield are observed for one batch transesterification by BCL-NKA in solvent-free and isooctane systems. However, considering the operational stability of lipase, it has been demonstrated that BCL-NKA-catalyzed transesterification for biodiesel production in the isooctane system is the optimum alternative. It is also suggested that there is a strong binding force between the BCL molecules and resin NKA support. Otherwise, it was impossible to keep a stable lipase activity to the next cycle. To further confirm the fastness between BCL and resin NKA, 20% Triton X-100 and SDS have been employed to treat the BCLNKA catalyst using ultrasonic assistance for 3 h. Then the concentration of protein in the supernatant is measured from 2.3% to 5.8%. Simultanuously, methanolysis of soybean oil using the supernatant as the catalyst was carried out to provide direct evidence that the supernatant seldom showed catalytic activity and the biodiesel yield is very low (