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Lipase-Mediated Selective Methanolysis of Fish Oil for Biodiesel Production and Polyunsaturated Fatty Acid Enrichment Gaojian Ma,† Lingmei Dai,† Dehua Liu,†,‡ and Wei Du*,†,‡ †
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Key Laboratory for Industrial Biocatalysis, Ministry of Education, Department of Chemical Engineering, Tsinghua University, Beijing 100084, People’s Republic of China ‡ Tsinghua Innovation Center in Dongguan, Dongguan, Guangdong 523808, People’s Republic of China ABSTRACT: Fish oil, containing quite a large amount of polyunsaturated fatty acids (PUFAs), is well-recognized as a good source for biodiesel and health care product production. A two-step process involving free lipase NS81006-mediated methanolysis followed by immobilized lipase Novozym 435-catalyzed conversion was proposed for the utilization of fish oil in this paper. During the lipase NS81006-catalzyed methanolysis process, the selectivity of lipase on different free fatty acids (FFAs) was studied systematically, and it was found that the length of the carbon chain and the number and position of C−C double bonds had a varied influence on the selectivity of lipase. The reaction rate and biodiesel yield decreased with the increase of the carbon chain length; higher conversion could be obtained with unsaturated FFAs (C18:1, C18:2, and C18:3) compared to unsaturated FFA (C18:0), among which FFA with three double bonds gave the highest conversion. Through this two-step lipase-mediated catalysis, both the conventional FFAs and PUFAs could be converted to their corresponding fatty acid methyl ester (FAME) effectively and a FAME yield of over 90% was obtained. The studies provide a rational guidance for biodiesel production as well as the enrichment of PUFAs. lipase.9,10 To eliminate the above-mentioned negative effect caused by byproduct glycerol during immobilized lipasemediated alcoholysis as well as considering the rather high cost of immobilized lipase, special interest has been paid to free lipase-mediated biodiesel production. Free lipase is a promising biocatalyst for biodiesel preparation as a result of its faster reaction rate, lower cost, and lower requirements on the feedstock, showing great prospect for practical biodiesel production.2,9,10 In this paper, we explored the potential of using free lipasecatalyzing fish oil for biodiesel preparation combined with PUFA enrichment. To realize the thorough conversion of FFA to its corresponding fatty acid methyl ester (FAME), a twostep process involving free lipase NS81006-mediated methanolysis of fish oil followed by the immobilized lipase Novozym 435-catalyzed methanolysis was proposed. During the process, the selectivity of lipase toward different FFAs was explored systematically.
1. INTRODUCTION Biodiesel has drawn increasing attention in recent decades as a renewable fuel and an ideal substitute for fossil diesel.1 The common preparation methods of biodiesel presently mainly include the chemical method, lipase method, and supercritical liquid method, among which the lipase method has received the most attention because of its mild reaction condition and low requirements to oil material.2−5 Different from other common oils, fish oil, such as tuna oil and sardine oil, contains a high content of long-chain free fatty acids (FFAs), especially a high concentration of polyunsaturated fatty acids (PUFAs), such as docosahexaenoic acid (DHA, C22:6), docosapentaenoic acid (DPA, C22:5), and eicosapentaenoic acid (EPA, C20:5), which have been proven to play a significant role in human health, such as the suitable development of the nervous system and the prevention of cardiovascular diseases.6 Many studies have focused on the enrichment of PUFAs from fish oils using lipases. He et al.7 reported that Candida antarctica lipase A (CAL-A) could concentrate ω-3 PUFAs from 25% in oils to nearly 90% in monoacylglycerols via one-step enzymatic ethanolysis. Haraldsson et al.8 tested 17 lipases for concentrating EPA and DHA from fish oil by lipase-catalyzed transesterification of fish oil with ethanol under anhydrous solvent-free conditions, and results showed that Pseudomonas lipases had the highest activity toward saturated and monounsaturated fatty acids in the fish oil, with much lower activity toward EPA and DHA. In general, immobilized lipase has been explored extensively for PUFA enrichment with fish oil as the feedstock as a result of its well-recognized advantage. However, it has been wellrecognized that, during immobilized lipase-catalyzed transesterification of the oil feedstock, byproduct glycerol had a serious negative effect on the catalytic performance of © XXXX American Chemical Society
2. MATERIALS AND METHODS 2.1. Chemicals and Lipases. The fish oil was kindly donated by Hai Zhiyuan Co., Ltd. (Guangzhou, China). Free lipase NS81006 (from Aspergillus niger, activity of 3300 units/mL) was a generous gift from Novo Industries (Denmark). Immobilized lipase Novozym 435 [from C. antarctica, activity of 10 000 propyl laurate units (PLU)/g] was obtained from Novozymes (Denmark). The standards of FAMEs (C14:0−C22:6) and heptadecanoic acid methyl ester as an internal gas chromatography (GC) standard as well as monoacylglycerides (MAGs), diacylglycerides (DAGs), and triacylglycerides (TAGs) as a high-performance liquid chromatography (HPLC) external standard Received: March 5, 2018 Revised: June 7, 2018 Published: June 12, 2018 A
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
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Energy & Fuels were purchased from Sigma-Aldrich (St. Louis, MO, U.S.A.). Other reagents of analytical grade were obtained commercially. 2.2. Sample Analysis. 2.2.1. Analysis of FFA Composition and Determination of the FAME Yield. The FFA composition of fish oil and the fixed FAME content are measured by the standard procedure AOAC 991.39. The detailed procedure is described as follows: weigh 25 mg of lipid in a tube with 2 mg of heptadecanoic acid methyl ester as the internal standard, then add 1.5 mL of 0.5 M sodium hydroxide in methanol, and heat for 15 min at 100 °C. After cooling, add 2 mL of 14% BF3 (w/v) in methanol and then heat for 30 min at 100 °C. After cooling to 30−40 °C, add 1 mL of hexane and then shake for 30 s. Then, add 5 mL of saturated NaCl solution and shake it until phase separation. Take the upper hexane layer out, and 1 μL of the sample was injected for GC analysis.11 The FAME content is measured by the following procedure: weigh 6−8 mg of oil sample obtained from the alcoholysis of fish oil and 0.6 mL of 0.591 g/L heptadecanoic acid methyl ester (internal standard) ethanol solution. The sample was mixed by a shaker, and then 1 μL was injected for GC analysis. GC analysis conditions: a flame ionization detector (FID, Agilent 7890A) and a column [CB-FFAP (0.32 mm × 2 m, Chromapack) DB-1 (0.25 mm × 15 m, J&W Scientific, Folsom, CA, U.S.A.)] were used to carry out the analysis. The initial column temperature was set at 180 °C and held for 0.5 min, then heated to 250 °C at the rate of 10 °C/min, and held for 6 min. The temperature of the detector and injector was set at 250 and 245 °C, respectively. The FAME yield is calculated according to the following formula: FAME yield (%) =
obtained by the external standard. The glyceride content (MAGs, DAGs, and TAGs) was calculated by the standard curve obtained by the external standard. The quantitative analysis was accomplished according to the standard lines of MAGs, DAGs, and TAGs established by an external method (Figure 1). 2.2.3. Determination of the Acid Value and Water Content. The acid value of the oil sample was determined according to the Chinese National Standard GB/T 5530-2005, and the water content was determined by the Karl Fischer moisture method according to the Chinese National Standard GB/T 26626-2011. 2.3. Lipase-Mediated Methanolysis of Oil. The free lipasecatalyzed process was carried out in a 500 mL three-neck roundbottom flask equipped with a mechanical stirrer at 1000 rpm and immersed in a thermostat water bath of 45 °C for 9 h. The reaction mixture consisted of oil (100 g), 10% water (w/w, oil), and 2% enzyme (w/w, oil),with methanol/oil of 5:1 (molar ratio, oil) (the molar mass of fish oil was calculated on the basis of the saponification value according to the Chinese National Standard GB/T 553-1995). The methanol adding strategy stepwise is described as follows: 25, 25, 25, and 25% of total methanol was added to the reaction mixture at 1 h interval from 0 to 3 h. Samples were taken from the reaction mixture at specified times and centrifuged to obtain the oil layer for GC analysis. The immobilized lipase-catalyzed process was carried out in a 50 mL conical flask and placed in a thermostatic shaking table at 45 °C and 180 rpm. The reaction mixture comprised crude biodiesel collected from the free lipase-catalyzed methanolysis (10 g), molecular sieves of type 3 Å (6 g), and 5% enzyme (w/w, oil), with the molar ratio of methanol/oil of 3:1 (here, oil refers to the rest of the remaining unconverted oil, calculated from the difference between the initial total oil and the oil already converted to FAME in the first step). The methanol adding strategy was the same as that in the free lipase-catalytic process. Samples were taken from the reaction mixture at specified times and then centrifuged at 60 °C and vacuum degree of 0.1 MPa to steam methanol and obtain the oil layer for GC analysis.
FAME content × 100% fixed FAME content
In the paper, the error values in each graph were different, despite the fact that the error bars looked small. Each sample was measured 3 times, and the error value (σ) was calculated according to the measured values (x1, x2, and x3) and the average value (x̅). 3
σ=
∑i = 1 (xi − x ̅ )2 3
3. RESULTS AND DISCUSSION 3.1. Properties of Fish Oil. The analysis of fish oil indicated that the acid value and saponification value of the fish oil were 0.64 and 186.1 mg of KOH/g, respectively, with the water content of 0.20% and the fixed FAME content of 80%. Over 99% of the glycerides existing in the fish oil were TAGs, and there was almost no MAGs and DAGs contained in the fish oil. The profile of FFA in fish oil was further characterized (Table 2), and it was found that there were over 14 kinds of FFA existing in the fish oil, among which over 20% were PUFAs (EPA, 8.25%; DHA, 14.23%). The FFA composition of fish oil is obviously different from conventional oils, such as soybean oil. 3.2. Free Lipase-Mediated Methanolysis of Fish Oil. First, immobilized lipase Novozym 435 was adopted for catalyzing the methanolysis of fish oil for biodiesel preparation and PUFA enrichment. It was found that the final FAME yield was only 75%, and similar results were also reported.8,9 Alternatively, free lipase NS81006 was explored for the catalysis of fish oil because our previous research demonstrated that free lipase NS81006 could catalyze the methanolysis of soybean oil effectively for biodiesel production.12 In the following study, free lipase NS81006-mediated methanolysis of fish oil was conducted. Interestingly, the final FAME yield of fish oil was significantly lower than that of soybean oil (Figure 2). Because of the different FFA profiles contained in soybean oil and fish oil, as shown in Table 2, the study of different conversions of FFAs to their corresponding FAMEs was further specifically investigated (Figure 3).
2.2.2. Analysis of Glyceride Composition of Oil by HPLC. The glycerides, including TAGs, DAGs, and MAGs, in the oil mixture were analyzed by a Shimadzu 20A HPLC system (Shimadzu Corp., Kyoto, Japan) equipped with an ELAD-LT II low-temperature evaporative light scattering detector. A C18 column (5 μm, 250 × 4.6 mm, PLATISIL ODS, Dikma Technology, China) was used for the separation at 40 °C. The mobile phase consisted of acetonitrile/acetic acid (99.85:0.15, %, v/v) and dichloromethane, which was pumped with a gradient elution program at the rate of 1.5 mL/min (Table 1). The drift pipe temperature was maintained at 40 °C, and the nitrogen pressure was controlled at 320 kPa. A total of 15 μL of sample and 1 mL of hexane were precisely measured and mixed thoroughly, and then 20 μL of the aforementioned mixture was injected for glyceride analysis. The glyceride content was calculated by the standard curve
Table 1. Gradient Elution Program of HPLC for Separating Glycerides time (min)
acetonitrile/acetic acid (99.85:0.15, %, v/v)
dichloromethane (%, v/v)
0 4 12 25 30 35 45 55 60 65
100 100 90 90 70 70 20 20 100 100
0 0 10 10 30 30 80 80 0 0 B
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
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Figure 1. External standard lines of MAGs, DAGs, and TAGs.
Table 2. FFA Composition of Fish Oil and Soybean Oil fatty acid
fish oil (wt %)
soybean oil (wt %)
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 C20:4 C20:5 C22:1 C22:6 other FFAs
5.89 18.2 6.43 3.62 14.5 2.90 1.74 4.57 1.04 8.25 5.74 14.2 12.9
nda 11.7 nd 4.33 24.6 51.7 6.49 nd nd nd nd nd nd
Figure 3. Conversion of different FFAs to their corresponding FAMEs.
a
nd = not detected.
The above results indicate that lipase NS81006 may have various selectivity toward different FFAs. To figure out the selectivity of lipase toward different FFAs, the length of the carbon chain and the number and position of C−C double bonds were considered and a further study on the conversion of particular FFAs to their corresponding FAMEs was systematically investigated. 3.2.1. Selectivity of Lipase toward FFAs with Different Lengths of the Carbon Chain. A longer chain of FFA means a higher steric hindrance, and it may hinder the combination of the ester bond of acylglycerol and the active site of lipase, resulting in the reduced conversion of FFA to its corresponding FAME.13 FAME yields of FFAs with different chain lengths (C14:0, C16:0, and C18:0) were compared to study the influence of the length of the carbon chain on the selectivity of lipase (Figure 4). As indicated in Figure 4, the reaction rate and FAME yield decrease with the increase of the length of the carbon chain, which matches with the steric hindrance theory. Concerning the tunnel-like binding pocket of lipase, the fatty acyl chain has to be introduced into the tunnel to realize the combination of
Figure 2. NS81006-mediated methanolysis of soybean oil and fish oil.
From Figure 3, it can be seen that the reaction rates of PUFAs (C20:4, C20:5, and C22:6) are slower than those of conventional FFAs, such as C18:0, C18:1, and C18:2. The FAME yield of DHA (C22:6) is only about 20%, while the FAME yields of other conventional FFAs reach about 80%. C
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
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methanolysis of fish oil, the double bond in FFAs would generally promote the conversion of FFAs to FAMEs. 3.2.3. Selectivity of Lipase on FFAs with Different Positions of the C−C Double Bond. PUFAs can be divided into two types according to the location of the C−C double bond that is the furthest away from the acyl group end, ω-3 PUFAs (double bond on the third carbon) and ω-6 PUFAs (double bond on the sixth carbon).16 Different locations of the double bond in the carbon chain usually mean different steric conformations of the FFA, hence affecting the steric hindrance and catalytic activity of lipase.17 From the comparison of the conversion of ω-3 PUFAs (C18:3, C20:5, and C22:6) and ω-6 PUFAs (C18:2 and C20:4) to their corresponding FAMEs, the selectivity of lipase toward FFAs with different positions of the C−C double bond was further investigated during NS81006-mediated methanolysis of fish oil. The catalytic activity of lipase on ω-6 PUFAs was much higher than that of ω-3 PUFAs (Figure 6), indicating that the steric hindrance of ω-3 PUFAs was higher than that of ω-6 PUFAs, which is in agreement with the results of the most relevant research.14
Figure 4. Conversion of FFAs with different lengths of chain to FAMEs.
the ester bond and the catalytic triad. This process would be more difficult for a longer fatty acyl chain, thus leading to the decrease of the catalytic activity of lipase toward FFAs having a longer acyl chain.13 3.2.2. Selectivity of Lipase on FFAs with Different Numbers of C−C Double Bonds. The catalytic activity of lipase toward different FFAs varies with the numbers of C−C double bonds.14 On one hand, the steric conformation of the carbon chain is stabilized because of the existence of the C−C double bond, which is beneficial for the combination of the ester bond and the active site of lipase. On the other hand, the existence of the C−C double bond promotes the intertangling of the carbon chain, increasing the steric hindrance and hindering the combination of the substitute and lipase.15 In the following study, conversion of FFAs of incremental number of C−C double bonds with the same chain length were investigated to study the influence of the number of C−C double bonds on the selectivity of lipase (panels a and b of Figure 5). Panels a and b of Figure 5 indicated that a higher FAME yield could be obtained with unsaturated FFAs (C16:1, C18:1, C18:2, and C18:3) compared to saturated FFAs (C16:0 and C18:0), and FFAs with more double bonds basically showed a higher FAME yield, although the FFAs of C18:1 and C18:2 showed almost the same FAME yield. The difference in catalyzing FFAs with various numbers of C−C double bonds may originate from the overlay of the positive and negative impacts of the C−C double bond on the steric hindrance of the carbon chain, and in the case of lipase NS81006-mediated
Figure 6. Conversion of FFAs with different positions of the double bond to FAMEs.
Among ω-3 PUFAs, EPA and DHA have been receiving the most attention as a result of their particular physiological effects on human health. Many studies have focused on the enrichment of EPA or DHA recently.18 Herein, it is of much significance to study the selectivity of lipase on EPA and DHA during methanolysis catalyzed by NS81006. From Figure 7, it could be noticed that the catalytic activity of NS81006 on EPA
Figure 5. Conversion of FFAs with different numbers of double bonds to FAMEs. D
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
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Figure 7. Conversion of EPA/DHA to FAMEs.
Figure 9. Second-step methanolysis catalyzed by Novozym 435 for FAME preparation.
is higher than that of DHA and about 80% of EPA was converted to its corresponding FAME, while only 20% of DHA was obtained (Figure 7). The ratio of conversion of EPA and DHA is a reflection of the selectivity of lipase toward DHA and EPA, and Figure 8 showed that the selectivity of NS81006 toward DHA and EPA maintained a high level, especially before 3 h, and reached the highest level at 2 h.
conversion of DHA to its corresponding methyl eaters (Figure 10).
Figure 10. Conversion of FFAs to FAMEs during Novozym 435mediated methanolysis.
Because the boiling point of PUFA methyl esters is higher than that of non-PUFAs (biodiesel, C14−C18), the separation of the biodiesel fraction and PUFA methyl esters could be simply achieved by vacuum distillation.9,19,20 Further, 100 g of oil obtained from the two-step conversion process was added to the vacuum distillation device, and the distillation was performed under pressure of 10−15 mbar. The fraction of 190−200 °C was collected as biodiesel. It was found that, in the biodiesel fraction, a FAME content of 97.3% could be obtained (the detailed component in this distillate is listed as Table 3) and the PUFA content in the residual oil phase was over 40%, including 29.0% DHA and 7.13% EPA. The rest of the PUFA methyl esters in the residual oil phase could be further evaporated by molecular distillation to obtain a higher PUFA concentration.19,20 Considering still, some amount of PUFA methyl esters remained in the distillate, which may influence the properties of biodiesel, such as its oxidation stability. Molecular distillation was further adopted for better separation of conventional methyl esters and PUFA methyl esters. It was found that by adopting molecular distillation (temperature, 80 °C; pressure, 2−5 Pa; feeding rate, 1 mL/min; and rotation speed, 200 rpm), a FAME content of 98.5% could be achieved, with the content of PUFA methyl esters lower than 4% in the distillate. The light phase can be used as biodiesel, and antioxidants or mixing with other FAMEs (such as biodiesel
Figure 8. Ratio of FAME yields of EPA and DHA.
The above study showed that lipase had varied selectivity toward different kinds of FFAs during the NS81006-mediated methanolysis of fish oil, which provides good guidance for biodiesel production and PUFA enrichment. After the first-step catalysis, characterization on the oil phase was further carried out and it was found that the acid value was 7.83 mg of KOH/g and MAGs and DAGs were 15.5 and 6.26%, respectively, while almost no TAGs existed in the reaction mixture. To further convert the rest of the FFAs, immobilized lipase Novozym 435 was adopted to catalyze the further methanolysis of fish oil after the free lipase-mediated process. 3.3. Second Step Catalyzed by Novozym 435 for Further Conversion of Fish Oil. The oil phase obtained from the free lipase-mediated process was separated by centrifugation and further reacted with methanol catalyzed by Novozym 435. The FAME yield increased significantly during Novozym 435-catalyzed methanolysis of fish oil in the second step, and a biodiesel yield of over 90% could be obtained (Figure 9). The conversion of different FFAs in the second step was further investigated, and it was found that the increase in the FAME yield in the second step was mainly due to the E
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
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(3) Su, E. Z.; Zhang, J. G.; Huang, M. G.; Wei, D. Z. Optimization of the lipase-catalyzed irreversible transesterification of Pistacia chinensis Bunge seed oil for biodiesel production. Russ. Chem. Bull. 2014, 63 (12), 2719−2728. (4) Su, E. Z.; Wei, D. Z. Production of Fatty Acid Butyl Esters Using the Low Cost Naturally Immobilized Carica papaya Lipase. J. Agric. Food Chem. 2014, 62 (27), 6375−6381. (5) Su, E. Z.; Wei, D. Z. Improvement in biodiesel production from soapstock oil by one-stage lipase catalyzed methanolysis. Energy Convers. Manage. 2014, 88, 60−65. (6) Bispo, P.; Batista, I.; Bernardino, R. J.; Bandarra, N. M. Preparation of Triacylglycerols Rich in Omega-3 Fatty Acids from Sardine Oil Using a Rhizomucor miehei Lipase: Focus in the EPA/ DHA Ratio. Appl. Biochem. Biotechnol. 2014, 172 (4), 1866−1881. (7) He, Y. J.; Li, J. B.; Kodali, S.; Chen, B.; Guo, Z. The near-ideal catalytic property of Candida antarctica lipase A to highly concentrate n-3 polyunsaturated fatty acids in monoacylglycerols via one-step ethanolysis of triacylglycerols. Bioresour. Technol. 2016, 219, 466− 478. (8) Haraldsson, G. G.; Kristinsson, B.; Sigurdardottir, R.; Gudmundsson, G. G.; Breivik, H. The Preparation of Concentrates of Eicosapentaenoic Acid and Docosahexaenoic Acid by LipaseCatalyzed Transesterification of Fish Oil with Ethanol. J. Am. Oil Chem. Soc. 1997, 74 (11), 1419−1424. (9) Tian, X. G.; Dai, L. M.; Liu, M. S.; Liu, D. H.; Du, W.; Wu, H. Lipase-catalyzed methanolysis of microalgae oil for biodiesel production and PUFAs concentration. Catal. Commun. 2016, 84, 44−47. (10) Lv, D.; Du, W.; Zhang, G. L.; Liu, D. H. Mechanism study on NS81006-mediated methanolysis of triglyceride in oil/water biphasic system for biodiesel production. Process Biochem. 2010, 45 (4), 446− 450. (11) Chen, J. Z.; Wang, S. M.; Zhou, B. Y.; Dai, L. M.; Liu, D. H.; Du, W. A robust process for lipase-mediated biodiesel production from microalgae lipid. RSC Adv. 2016, 6 (54), 48515−48522. (12) Lü, L. L.; Du, W.; Liu, D. H. Kinetics of Liquid Lipase NS81006-Catalyzed Alcoholysis of Oil for Biodiesel Production. Cuihua Xuebao 2012, 33 (11), 1857−1861. (13) Li, L. F.; Tang, Q. L.; Jiang, M. Z.; Jiang, S. Z. Enrichment of DHA and EPA glycerides by selective hydrolysis of waste fish oil with immobilized Geotrichun sp. lipase. Chin. J. Bioprocess Eng. 2009, 7 (6), 25−30. (14) Wanasundara, U. N.; Shahidi, F. Concentration of ω-3 Polyunsaturated Fatty Acids of Marine Oils Using Candida Cylindracea Lipase: Optimization of Reaction Conditions. J. Am. Oil Chem. Soc. 1998, 75 (12), 1767−1774. (15) Casas-Godoy, L.; Meunchan, M.; Cot, M.; Duquesne, S.; Bordes, F.; Marty, A. Yarrowia lipolytica lipase Lip2: An efficient enzyme for the production of concentrates of docosahexaenoic acid ethyl ester. J. Biotechnol. 2014, 180, 30−36. (16) Tian, X. G.; Du, W.; Dai, L. M.; Liu, D. H. The Development of Enzymatic Enrichment and Separation of ω-3PUFAs. J. Chem. Eng. Chin. Univ. 2015, 29 (6), 1285−1292. (17) Akanbi, T. O.; Barrow, C. J. Candida antarctica lipase A effectively concentrates DHA from fish and thraustochytrid oils. Food Chem. 2017, 229, 509−516. (18) Lee, E. J.; Lee, M. W.; No, D. S.; Kim, H. J.; Oh, S. W.; Kim, Y.; Kim, I. H. Preparation of high purity docosahexaenoic acid from microalgae oil in a packed bed reactor via two-step lipase-catalysed esterification. J. Funct. Foods 2016, 21, 330−337. (19) Privett, O. S.; Nadenicek, J. D.; Pusch, F. J.; Nickell, E. C. Application of High Vacuum Fractional Distillation to Complex Mixtures of Methyl Esters of Polyunsaturated Fatty Acids. J. Am. Oil Chem. Soc. 1969, 46 (1), 13−17. (20) Torres, S.; Acien, G.; García-Cuadra, F.; Navia, R. Direct transesterification of microalgae biomass and biodiesel refining with vacuum distillation. Algal Res. 2017, 28, 30−38.
Table 3. Contents of FAMEs in the Distillate fatty acid
FAME content (%)
C14:0 C16:0 C16:1 C18:0 C18:1 C18:2 C18:3 C20:1 C20:4 C20:5 C22:1 C22:6 other FFAs
6.90 22.0 9.20 3.69 19.1 3.59 2.45 4.61 0.98 7.64 5.43 1.81 9.9
from palm oil, which has a higher oxidation stability) can solve the oxidation stability issue caused by the existence of some minor PUFA methyl esters.
4. CONCLUSION It was found that only using a one-step lipase-catalyzed conversion could not realize the thorough conversion of fish oil for the synthesis of FAMEs catalyzed by either Novozym 435 or NS81006. A two-step process involving free lipase NS81006-mediated methanolysis followed by immobilized lipase Novozym 435-catalyzed conversion was applied for the preparation of biodiesel and enrichment of PUFAs, and a FAME yield of over 90% could be obtained. The selectivity of lipase toward different FFAs was studied systematically, and the results demonstrated that the length of th ecarbon chain and the number and position of the C−C double bonds had a varied influence on the selectivity of lipase. This process was especially suitable for the catalysis of non-edible oil feedstocks for the synthesis of FAMEs, especially for those containing complicated components, because, in the first step, free lipase costs much lower than the immobilized lipase and the corresponding risk could be reduced significantly.
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Wei Du: 0000-0003-4094-210X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors express their gratitude for the support from the Science and Technology Department of Guangdong Province (2015B090901006) and the Dongguan Science and Technology Bureau (Innovative R&D Team Leadership of Dongguan, 201536000100033).
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REFERENCES
(1) Chen, X.; Du, W.; Liu, D. H. Effect of several factors on soluble lipase-mediated biodiesel preparation in the biphasic aqueous-oil systems. World J. Microbiol. Biotechnol. 2008, 24 (10), 2097−2102. (2) Li, Y.; Du, W.; Dai, L. M.; Liu, D. H. Kinetic study on free lipase NS81006-catalyzed biodiesel production from soybean oil. J. Mol. Catal. B: Enzym. 2015, 121, 22−27. F
DOI: 10.1021/acs.energyfuels.8b00749 Energy Fuels XXXX, XXX, XXX−XXX
