Bioresource Technology 174 (2014) 302–305
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodiesel production by combined fatty acids separation and subsequently enzymatic esterification to improve the low temperature properties Meng Wang a, Kaili Nie a,b, Hao Cao a, Li Deng a,b,⇑, Fang Wang a,c, Tianwei Tan a a b c
Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China Amoy – BUCT Industrial Bio-Technovation Institute, Amoy 361022, PR China State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Design of a novel process for
Waste oil hydrolysis, urea complexation and enzymatic esterification are combined to produce biodiesel with improved CFPP.
enzymatic synthesis of biodiesel. Eliminate low temperature crystallization. Waste oil hydrolysis, urea complexation and enzymatic esterification are combined. Low temperature properties were considerably improved through the novel process.
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 1 August 2014 Accepted 2 August 2014 Available online 9 August 2014 Keywords: Biodiesel Hydrolysis Urea complexation Esterification Cold filter plugging point
a b s t r a c t The poor low-temperature properties of biodiesel, which provokes easy crystallization at low temperature, can cause fuel line plugging and limits its blending amount with petro-diesel. This work aimed to study the production of biodiesel with a new process of improving the low temperature performance of biodiesel. Waste cooking oil was first hydrolyzed into fatty acids (FAs) by 60 g immobilized lipase and 240 g RO water in 15 h. Then, urea complexation was used to divide the FAs into saturated and unsaturated components. The conditions for complexation were: FA-to-urea ratio 1:2 (w/w), methanol to FA ratio 5:1 (v/v), duration 2 h. The saturated and unsaturated FAs were then converted to iso-propyl and methyl esters by lipase, respectively. Finally, the esters were mixed together. The CFPP of this mixture was decreased from 5 °C to 3 °C. Hydrolysis, urea complexation and enzymic catalyzed esterification processes are discussed in this paper. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, usually considered as fatty acid methyl esters (FAMEs), is an alternative fuel for diesel engines. It had been tested as an alternative fuel source since the energy crisis in the 1970s. ⇑ Corresponding author at: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China. Tel.: +86 010 64451636; fax: +86 010 64416428. E-mail address: [email protected] (L. Deng). http://dx.doi.org/10.1016/j.biortech.2014.08.011 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Biodiesel is attractive because it is a biodegradable and renewable energy source with higher flash point and excellent lubricity. The environmental benefits of using biodiesel include lower exhaust emissions of particulate matter, CO and SOx (Jiang et al., 2014; Jo et al., 2014; Kuo et al., 2013). However, the main disadvantage, limiting the upper fraction of blending biodiesel with petro-diesel at 20% or less, is its relatively poor low-temperature properties (Dunn et al., 1996; Smith et al., 2010). Especially in winter, when the temperature falls below 0 °C, the saturated methyl ester of waste oil will nucleate and form crystals, that propressively plug
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or restrict flow through fuel lines and filters during engine start-up can lead to fuel starvation and engine failure (Knothe et al., 2005). However, the problems of cold plugging are not only experienced at cold start-up, but also during the use of the engine if the temperature suddenly starts to drop. As was reported, the unsaturated FAMEs have good low-temperature properties, but poor oxidative stability and low cetane numbers (Chastek, 2011; Knothe, 2005; Smith et al., 2010). The saturated FAMEs have good oxidative stability and high cetane numbers, and could increase the cold filter plugging point (CFPP) (ASTM, 2010). However, ethyl and iso-propyl esters have improved low-temperature properties without reducing cetane number or oxidative stability (Knothe, 2008; Lee et al., 1995). Taking a typical saturated fatty acid–palmitic acid as an example, the melting point of its methyl, ethyl- and i-propyl esters are 30.5 °C, 24 °C, 13–14 °C, respectively (Knothe et al., 2005). As shown in the results, the melting point of iso-propyl ester of palmitic acid decreased significantly when compared with methyl esters. The sole esterification with ethanol and 1-propanol is however economically prohibitive against the use of cheap methanol. In this study, waste cooking oil (WCO) was used as raw material to prepare biodiesel. In contrast to previous process, WO was first hydrolyzed into free fatty acid, and then urea complexation was used to separate free fatty acid into saturated and unsaturated fractions. Using Candida sp. 99–125 lipase for catalyst, the unsaturated and saturated free fatty acids were converted to methyl and iso-propyl esters respectively (Hama et al., 2013; Lu et al., 2008). The mix of these two esters obviously improved the CFPP of FAMEs from WO. The processes of WO hydrolysis, urea complexation and enzymic catalyzed esterification will be discussed.
2.3. FA separation via urea complexation Methanol, urea and FAME were mixed into a three neck flask with a reflux and mechanical stirring at a reaction temperature of 50 °C and stirring time 50 min (Bi et al., 2010). Then the mixture was cooled to a specific temperature of 20 °C for a specific time of 2 h. The urea complexes (crystals) and non-urea complexes (filtrate) were separated by filtration with a Buchner funnel. The methanol was recovered from the filtrate by a rotary evaporator under vacuum. The filtrate was washed with saturated sodium chloride solution in 60 °C for two times and then was washed with distilled water in 80–85 °C for one time to remove residual methanol and urea (Stephen et al., 2006). Finally, the unsaturated FA with a low melting point from filtrate was obtained by removing the residue water through a rotary evaporator at 90 °C under vacuum (residue pressure 200 Pa) for 1 h. The urea complexes were dissolved in distilled water under the condition of 5:8 (w/w) of water to complexes ratio at room temperature, and then the FA in the non-aqueous phase (top layer) was separated from the aqueous phase by a separating funnel. After washing steps as applied for the above filtrate, the FA with a high-melting-point from urea complexes was obtained by removing residual water with a rotary evaporator. The FA yield of non-urea complexes and urea complexes was calculated as the FA weight of non-complexes and urea complexes divided by the total weight of FA before urea complexation, respectively, as shown in Eqs. (1) and (2) (Bi et al., 2010).
Yield of non-urea complexes ð%Þ ¼
2. Methods 2.1. Materials Waste cooking oil was obtained from Lvming Co. Ltd. Shanghai, China. The waste cooking oil (WCO) contained 83.9% of free fatty acids (FFAs), 0.5% of monoacylglycerols (MAGs), 6.9% of diacylglycerols (DAGs), and 8.7% of triacylglycerols (TAGs). The FFA composition of WCO was as following (wt%): C14:0, 0.8%; C16:0, 21.2%; C16:1, 1.2%; C18:0, 6.6%; C18:1, 34.6%; C18:2, 30.6%; C20:0, 1.0%; C22:1, 0.6%. Based on the FFA composition, the average molecular weight of FFAs was measured at 275.9 g/mol. Free lipase from Candida sp. 99–125 was obtained from Kaitai Biochemical Technology Company, Beijing, China; and the activity of the enzyme powder was 50,000 U/g. Other chemicals used in this paper were analytical grade and obtained from Beijing Chemical Factory, Beijing, China. 2.2. Hydrolysis of WO by lipase The hydrolysis was carried out in a 1 L triple-neck flask with constant agitation at 40 °C. The reaction system contained 200 g WO, 60 g immobilized lipase and 240 g RO water, with 16 h reaction time in total. For analysis, 0.5 ml mixture was taken for acid value determination every hour. For gas chromatography analysis, another 200 ll mixture was taken and centrifuged to harvest the supernatant or upper layer. Then 10 ll of the supernatant was dissolved in n-hexane for gas chromatography analysis. All the experiments were replicated at least three times and the results presented were the mean values for the replicated data. The error bars are presented in the figures. Finally, the residual water from the upper layer of the centrifuged reaction mixture was removed by distillation, leaving a 100% oil phase.
Yield of urea complexes ð%Þ ¼
Non-urea complexes of FA 100% FA before cystallisation ð1Þ
Urea complexes of FA 100% FA before crystallization ð2Þ
where the non-urea complexes of FA is the weight of FA as filtrate, the urea complexes of FA is the weight of FA obtained as crystals, and the FA before crystallization is the weight of FA added into flask.
2.4. Gas chromatography analysis The free fatty acid and esters contents in the mixture were quantified by a GC-2010 gas chromatography (GC, Shimadzu Japan). The GC analytical method was the same as reported (Liu et al., 2014). Heptadecanoic acid and its methyl ester purchased from Sigma were used as an internal standard. The esters0 yield is defined as esters amount produced divided by the initial amount of oil (g/g).
2.5. Enzyme catalyzed esterification The methanolysis and iso-propanolysis were carried out in a 1 L triple-neck flask with constant agitation at 40 °C. The reaction system contains 600 g waste oil, 80 g immobilized lipase and 60 g RO water. The reaction time was in total 40 h, and 4.6 g methanol or 8.6 g iso-propanol was added into the reaction system every 2 h (Lu et al., 2010). For analysis, 0.5 ml mixture was taken for acid value determination every hour. The analytical procedure was identical as described in Section 2.2.
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2.6. Determination of cold filter plugging point CFPP were determined by a multifunction determinator (FCJH205), using a standard method ASTM 6371-05 as described previously. 3. Results and discussion 3.1. Hydrolysis reaction process As the WCO was partly hydrolyzed during cooking, collecting and transportation, the WCO used in experiment had a relatively high content of fatty acids as shown in Section 2.1. Immobilized Candida sp. 99–125 lipase, which could catalyze both hydrolysis and esterification reaction (Lu et al., 2008), was used as catalyst of WCO hydrolysis. From 0 h to 8 h, the hydrolysis rate was quite fast because there was less glycerol which was product of this hydrolytic reaction. However, from 8 h to 15 h, the rate became slow, and the FA yields reached up to 99.2% at 15 h. After that, the FA yields became stable because of reaction balance as shown in Fig. 1, which is caused by the properties of lipase. Therefore, to obtain more FA for esterification, 15 h was chosen as the optimal hydrolysis time. 3.2. Effect of FA-to-urea ratio on unsaturated fatty acids yields
unsaturated FA yields as measured in the filtrate after 2 h were recorded. The unsaturated FA yields increased significantly from 70.4% to 87.4% as the ratio of FA-to-urea increased from 1:0 to 1:2. However, as the ratio of FA-to-urea increased from 1:2 to 1:3, the unsaturated FA yields just had a slight increase. This result indicated that a FA-to-urea ratio of 1:2 could provide enough crystalline substance. Taking the production costs and unsaturated FA yields into consideration, the FA-to-urea ratio 1:2 was selected as optimal condition.
3.3. Effect of FA-to-methanol ratio on unsaturated fatty acids yields In urea complexation, methanol was used as a solvent to dissolve the urea and fatty acids so that the urea and fatty acid could contact well and provide an appropriate crystalline environment. Since entrained methanol in the urea complexes was almost constant, when the methanol amount was increased, unsaturated fatty acids dissolved in the entrained methanol would be reduced, and the unsaturated fatty acids yield would be improved, as shown in Fig. 2. On the other hand, when the amount of methanol increased, more urea was dissolved, so it became more difficult to crystallize. However, as the solubility of urea in methanol was limited at low temperature, the unsaturated fatty acids’ content reduced slightly, as shown in Fig. 2. As amounts of methanol in excess of 5–6, the unsaturated fatty acids’ yields remain nearly constant because of good decentralization and less entrained unsaturated fatty acids. Considering the unsaturated fatty acid content and the filtrate yield, the methanol to fatty acids ratio 5 was selected as the optimal reaction condition.
The amount of urea added in the urea complexation process is an important parameter. Since urea is the primary crystalline substance, the amount of urea influences the amount of the complexation of saturated fatty acids. In the crystallization process, the urea crystal can form a hexagonal-hollow channel structure of 8.23 Å width (between the two sides), and can hold a saturated linear hydrocarbon chain with the cross-sectional size of 7.48 Å 4.99 Å (Smith, 1952), which facilitates the penetration of saturated fatty acids, that hence remain associated with the solid phase. However, the non-rotational double bonds in unsaturated fatty acids turn even the linear hydrocarbon chain into a cross-sectional size over 8.23 Å. This steric hindrance opposes unsaturated fatty acids0 penetration, leaving them in the methanol solution. The complexations were performed for varying FA-to-urea ratio, ranging from 1:0 to 1:3 (w/w) at temperature of 50 °C and stirring time 50 min. The mixture was cooled to a specific temperature of 20 °C. The
As the production process of methyl esters is mature, the reported method was used for methanolysis (Liu et al., 2014; Nie et al., 2006), and immobilized Candida sp. 99–125 was used as catalyst of WCO methanolysis. But for enzymatic isopropanolysis, there was no reported method, so the same method as methanolysis was tried. As shown in Fig. 1, from 0 h to 24 h, the isopropanolysis rate was quite fast. However, from 24 h to 40 h, the rate became slow, which indicated esterification and hydrolysis reaction rates reached a balance. At 40 h, the isopropyl esters yields
Fig. 1. Enzymatic hydrolysis process of waste cooking oil and isopropanolysis reaction course (Section 2.2 and 2.5). The fatty acids0 yields and isopropyl esters0 yields, respectively (4, h).
Fig. 2. The influence of methanol-to-FA ratio. The unsaturated fatty acids0 content and unsaturated fatty acids0 yields, respectively (j, d).
3.4. Enzyme catalyzed esterification of saturated fatty acids and isopropanol
M. Wang et al. / Bioresource Technology 174 (2014) 302–305
reached up to 98.9%. Therefore, 40 h was chosen as optimal isopropanolysis time.
3.5. The CFPP of the ester mixture Finally, the two esters, methyl unsaturated fatty acid esters and isopropyl saturated fatty acid esters, were mixed together. The CFPP (cold filter plugging point) of this mixture was measured, and the CFPP of this mixture decreased from 5 °C to 3 °C. This decrease was mainly contributed by the isopropyl saturated fatty acid ester. When the saturated fatty acids were turned to methyl esters, they had relatively high freezing point and made the biodiesel easy to crystallize (CFPP was 5 °C). However, once they were turned to isopropyl esters and had dendritic head structure, which differed from FA methyl esters, crystallization in the mixture became more difficult (CFPP was 3 °C). Therefore, the biodiesel which was composed of methyl and isopropyl esters had a lower CFPP than the whole methyl biodiesel.
4. Conclusions The new process of improving the low temperature performance of biodiesel had a significant effect. The CFPP (cold filter plugging point) of the esters mixture was decreased from 5 °C to 3 °C. This process was different from winterizing FAME. In this process, all the fatty acids are consumed, and the whole process did not have any material losses. Furthermore, using lipase, an environmentally friendly catalyst, greatly reduces the environmental pollution in comparison with the commonly used chemical catalysts.
Acknowledgements This research was financially supported by the Key Projects in the National Science & Technology Pillar Program during the 12th Five-year Plan Period (No. 2011BAD22B04), Amoy Industrial Biotechnology R&D and Pilot Conversion Platform (No. 3502Z20121009), the Fundamental Research Funds for the Central University (ZY1331), the Transformation of Agricultural Science and Technology Achievements Project (2013GB2C410539).
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References Akers, Stephen M., Thomas, J.L.C. Stephanie N., Rider, Keith B., 2006. Determination of the heat of combustion of biodiesel using bomb calorimetry. A multidisciplinary undergraduate chemistry experiment. J. Chem. Educ. 83 (2), 260–262. ASTM, 2010. Standard test method for cold filter plugging point of diesel and heating fuels, vol. 6371-05. http://www.astm.org/Standards/D6371.htm. Bi, Y., Ding, D., Wang, D., 2010. Low-melting-point biodiesel derived from corn oil via urea complexation. Bioresour. Technol. 101 (4), 1220–1226. Chastek, T.Q., 2011. Improving cold flow properties of canola-based biodiesel. Biomass Bioenergy 35 (1), 600–607. Dunn, R., Shockley, M., Bagby, M., 1996. Improving the low-temperature properties of alternative diesel fuels: vegetable oil-derived methyl esters. J. Am. Oil Chem. Soc. 73 (12), 1719–1728. Hama, S., Yoshida, A., Tamadani, N., Noda, H., Kondo, A., 2013. Enzymatic production of biodiesel from waste cooking oil in a packed-bed reactor: an engineering approach to separation of hydrophilic impurities. Bioresour. Technol. 135, 417– 421. Jiang, Y., Liu, X., Chen, Y., Zhou, L., He, Y., Ma, L., Gao, J., 2014. Pickering emulsion stabilized by lipase-containing periodic mesoporous organosilica particles: a robust biocatalyst system for biodiesel production. Bioresour. Technol. 153, 278–283. Jo, Y.J., Lee, O.K., Lee, E.Y., 2014. Dimethyl carbonate-mediated lipid extraction and lipase-catalyzed in situ transesterification for simultaneous preparation of fatty acid methyl esters and glycerol carbonate from Chlorella sp. KR-1 biomass. Bioresour. Technol. 158, 105–110. Knothe, G., 2005. Dependence of biodiesel fuel properties on the structure of fatty acid alkyl esters. Fuel Process. Technol. 86 (10), 1059–1070. Knothe, G., 2008. ‘‘Designer’’ biodiesel: optimizing fatty ester composition to improve fuel properties. Energy Fuels 22 (2), 1358–1364. Knothe, G., Van Gerpen, J.H., Krahl, J., 2005. The Biodiesel Handbook. AOCS Press Champaign, IL. Kuo, C.-H., Peng, L.-T., Kan, S.-C., Liu, Y.-C., Shieh, C.-J., 2013. Lipase-immobilized biocatalytic membranes for biodiesel production. Bioresour. Technol. 145, 229– 232. Lee, I., Johnson, L.A., Hammond, E.G., 1995. Use of branched-chain esters to reduce the crystallization temperature of biodiesel. J. Am. Oil. Chem. Soc. 72 (10), 1155–1160. Liu, S., Nie, K., Zhang, X., Wang, M., Deng, L., Ye, X., Wang, F., Tan, T., 2014. Kinetic study on lipase-catalyzed biodiesel production from waste cooking oil. J. Mol. Catal. B Enzym. 99, 43–50. Lu, J., Deng, L., Zhao, R., Zhang, R., Wang, F., Tan, T., 2010. Pretreatment of immobilized Candida sp. 99–125 lipase to improve its methanol tolerance for biodiesel production. J. Mol. Catal. B Enzym. 62 (1), 15–18. Lu, J., Nie, K., Wang, F., Tan, T., 2008. Immobilized lipase Candida sp. 99–125 catalyzed methanolysis of glycerol trioleate: solvent effect. Bioresour. Technol. 99 (14), 6070–6074. Nie, K., Xie, F., Wang, F., Tan, T., 2006. Lipase catalyzed methanolysis to produce biodiesel: optimization of the biodiesel production. J. Mol. Catal. B Enzym. 43 (1–4), 142–147. Smith, A., 1952. The crystal structure of the urea-hydrocarbon complexes. Acta Crystallogr. A 5 (2), 224–235. Smith, P.C., Ngothai, Y., Dzuy Nguyen, Q., O’Neill, B.K., 2010. Improving the lowtemperature properties of biodiesel: methods and consequences. Renewable Energy 35 (6), 1145–1151.
Contents lists available at ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Biodiesel production by combined fatty acids separation and subsequently enzymatic esterification to improve the low temperature properties Meng Wang a, Kaili Nie a,b, Hao Cao a, Li Deng a,b,⇑, Fang Wang a,c, Tianwei Tan a a b c
Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China Amoy – BUCT Industrial Bio-Technovation Institute, Amoy 361022, PR China State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, PR China
h i g h l i g h t s
g r a p h i c a l a b s t r a c t
Design of a novel process for
Waste oil hydrolysis, urea complexation and enzymatic esterification are combined to produce biodiesel with improved CFPP.
enzymatic synthesis of biodiesel. Eliminate low temperature crystallization. Waste oil hydrolysis, urea complexation and enzymatic esterification are combined. Low temperature properties were considerably improved through the novel process.
a r t i c l e
i n f o
Article history: Received 20 June 2014 Received in revised form 1 August 2014 Accepted 2 August 2014 Available online 9 August 2014 Keywords: Biodiesel Hydrolysis Urea complexation Esterification Cold filter plugging point
a b s t r a c t The poor low-temperature properties of biodiesel, which provokes easy crystallization at low temperature, can cause fuel line plugging and limits its blending amount with petro-diesel. This work aimed to study the production of biodiesel with a new process of improving the low temperature performance of biodiesel. Waste cooking oil was first hydrolyzed into fatty acids (FAs) by 60 g immobilized lipase and 240 g RO water in 15 h. Then, urea complexation was used to divide the FAs into saturated and unsaturated components. The conditions for complexation were: FA-to-urea ratio 1:2 (w/w), methanol to FA ratio 5:1 (v/v), duration 2 h. The saturated and unsaturated FAs were then converted to iso-propyl and methyl esters by lipase, respectively. Finally, the esters were mixed together. The CFPP of this mixture was decreased from 5 °C to 3 °C. Hydrolysis, urea complexation and enzymic catalyzed esterification processes are discussed in this paper. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel, usually considered as fatty acid methyl esters (FAMEs), is an alternative fuel for diesel engines. It had been tested as an alternative fuel source since the energy crisis in the 1970s. ⇑ Corresponding author at: Beijing Bioprocess Key Laboratory, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing, 100029, PR China. Tel.: +86 010 64451636; fax: +86 010 64416428. E-mail address: [email protected] (L. Deng). http://dx.doi.org/10.1016/j.biortech.2014.08.011 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
Biodiesel is attractive because it is a biodegradable and renewable energy source with higher flash point and excellent lubricity. The environmental benefits of using biodiesel include lower exhaust emissions of particulate matter, CO and SOx (Jiang et al., 2014; Jo et al., 2014; Kuo et al., 2013). However, the main disadvantage, limiting the upper fraction of blending biodiesel with petro-diesel at 20% or less, is its relatively poor low-temperature properties (Dunn et al., 1996; Smith et al., 2010). Especially in winter, when the temperature falls below 0 °C, the saturated methyl ester of waste oil will nucleate and form crystals, that propressively plug
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or restrict flow through fuel lines and filters during engine start-up can lead to fuel starvation and engine failure (Knothe et al., 2005). However, the problems of cold plugging are not only experienced at cold start-up, but also during the use of the engine if the temperature suddenly starts to drop. As was reported, the unsaturated FAMEs have good low-temperature properties, but poor oxidative stability and low cetane numbers (Chastek, 2011; Knothe, 2005; Smith et al., 2010). The saturated FAMEs have good oxidative stability and high cetane numbers, and could increase the cold filter plugging point (CFPP) (ASTM, 2010). However, ethyl and iso-propyl esters have improved low-temperature properties without reducing cetane number or oxidative stability (Knothe, 2008; Lee et al., 1995). Taking a typical saturated fatty acid–palmitic acid as an example, the melting point of its methyl, ethyl- and i-propyl esters are 30.5 °C, 24 °C, 13–14 °C, respectively (Knothe et al., 2005). As shown in the results, the melting point of iso-propyl ester of palmitic acid decreased significantly when compared with methyl esters. The sole esterification with ethanol and 1-propanol is however economically prohibitive against the use of cheap methanol. In this study, waste cooking oil (WCO) was used as raw material to prepare biodiesel. In contrast to previous process, WO was first hydrolyzed into free fatty acid, and then urea complexation was used to separate free fatty acid into saturated and unsaturated fractions. Using Candida sp. 99–125 lipase for catalyst, the unsaturated and saturated free fatty acids were converted to methyl and iso-propyl esters respectively (Hama et al., 2013; Lu et al., 2008). The mix of these two esters obviously improved the CFPP of FAMEs from WO. The processes of WO hydrolysis, urea complexation and enzymic catalyzed esterification will be discussed.
2.3. FA separation via urea complexation Methanol, urea and FAME were mixed into a three neck flask with a reflux and mechanical stirring at a reaction temperature of 50 °C and stirring time 50 min (Bi et al., 2010). Then the mixture was cooled to a specific temperature of 20 °C for a specific time of 2 h. The urea complexes (crystals) and non-urea complexes (filtrate) were separated by filtration with a Buchner funnel. The methanol was recovered from the filtrate by a rotary evaporator under vacuum. The filtrate was washed with saturated sodium chloride solution in 60 °C for two times and then was washed with distilled water in 80–85 °C for one time to remove residual methanol and urea (Stephen et al., 2006). Finally, the unsaturated FA with a low melting point from filtrate was obtained by removing the residue water through a rotary evaporator at 90 °C under vacuum (residue pressure 200 Pa) for 1 h. The urea complexes were dissolved in distilled water under the condition of 5:8 (w/w) of water to complexes ratio at room temperature, and then the FA in the non-aqueous phase (top layer) was separated from the aqueous phase by a separating funnel. After washing steps as applied for the above filtrate, the FA with a high-melting-point from urea complexes was obtained by removing residual water with a rotary evaporator. The FA yield of non-urea complexes and urea complexes was calculated as the FA weight of non-complexes and urea complexes divided by the total weight of FA before urea complexation, respectively, as shown in Eqs. (1) and (2) (Bi et al., 2010).
Yield of non-urea complexes ð%Þ ¼
2. Methods 2.1. Materials Waste cooking oil was obtained from Lvming Co. Ltd. Shanghai, China. The waste cooking oil (WCO) contained 83.9% of free fatty acids (FFAs), 0.5% of monoacylglycerols (MAGs), 6.9% of diacylglycerols (DAGs), and 8.7% of triacylglycerols (TAGs). The FFA composition of WCO was as following (wt%): C14:0, 0.8%; C16:0, 21.2%; C16:1, 1.2%; C18:0, 6.6%; C18:1, 34.6%; C18:2, 30.6%; C20:0, 1.0%; C22:1, 0.6%. Based on the FFA composition, the average molecular weight of FFAs was measured at 275.9 g/mol. Free lipase from Candida sp. 99–125 was obtained from Kaitai Biochemical Technology Company, Beijing, China; and the activity of the enzyme powder was 50,000 U/g. Other chemicals used in this paper were analytical grade and obtained from Beijing Chemical Factory, Beijing, China. 2.2. Hydrolysis of WO by lipase The hydrolysis was carried out in a 1 L triple-neck flask with constant agitation at 40 °C. The reaction system contained 200 g WO, 60 g immobilized lipase and 240 g RO water, with 16 h reaction time in total. For analysis, 0.5 ml mixture was taken for acid value determination every hour. For gas chromatography analysis, another 200 ll mixture was taken and centrifuged to harvest the supernatant or upper layer. Then 10 ll of the supernatant was dissolved in n-hexane for gas chromatography analysis. All the experiments were replicated at least three times and the results presented were the mean values for the replicated data. The error bars are presented in the figures. Finally, the residual water from the upper layer of the centrifuged reaction mixture was removed by distillation, leaving a 100% oil phase.
Yield of urea complexes ð%Þ ¼
Non-urea complexes of FA 100% FA before cystallisation ð1Þ
Urea complexes of FA 100% FA before crystallization ð2Þ
where the non-urea complexes of FA is the weight of FA as filtrate, the urea complexes of FA is the weight of FA obtained as crystals, and the FA before crystallization is the weight of FA added into flask.
2.4. Gas chromatography analysis The free fatty acid and esters contents in the mixture were quantified by a GC-2010 gas chromatography (GC, Shimadzu Japan). The GC analytical method was the same as reported (Liu et al., 2014). Heptadecanoic acid and its methyl ester purchased from Sigma were used as an internal standard. The esters0 yield is defined as esters amount produced divided by the initial amount of oil (g/g).
2.5. Enzyme catalyzed esterification The methanolysis and iso-propanolysis were carried out in a 1 L triple-neck flask with constant agitation at 40 °C. The reaction system contains 600 g waste oil, 80 g immobilized lipase and 60 g RO water. The reaction time was in total 40 h, and 4.6 g methanol or 8.6 g iso-propanol was added into the reaction system every 2 h (Lu et al., 2010). For analysis, 0.5 ml mixture was taken for acid value determination every hour. The analytical procedure was identical as described in Section 2.2.
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2.6. Determination of cold filter plugging point CFPP were determined by a multifunction determinator (FCJH205), using a standard method ASTM 6371-05 as described previously. 3. Results and discussion 3.1. Hydrolysis reaction process As the WCO was partly hydrolyzed during cooking, collecting and transportation, the WCO used in experiment had a relatively high content of fatty acids as shown in Section 2.1. Immobilized Candida sp. 99–125 lipase, which could catalyze both hydrolysis and esterification reaction (Lu et al., 2008), was used as catalyst of WCO hydrolysis. From 0 h to 8 h, the hydrolysis rate was quite fast because there was less glycerol which was product of this hydrolytic reaction. However, from 8 h to 15 h, the rate became slow, and the FA yields reached up to 99.2% at 15 h. After that, the FA yields became stable because of reaction balance as shown in Fig. 1, which is caused by the properties of lipase. Therefore, to obtain more FA for esterification, 15 h was chosen as the optimal hydrolysis time. 3.2. Effect of FA-to-urea ratio on unsaturated fatty acids yields
unsaturated FA yields as measured in the filtrate after 2 h were recorded. The unsaturated FA yields increased significantly from 70.4% to 87.4% as the ratio of FA-to-urea increased from 1:0 to 1:2. However, as the ratio of FA-to-urea increased from 1:2 to 1:3, the unsaturated FA yields just had a slight increase. This result indicated that a FA-to-urea ratio of 1:2 could provide enough crystalline substance. Taking the production costs and unsaturated FA yields into consideration, the FA-to-urea ratio 1:2 was selected as optimal condition.
3.3. Effect of FA-to-methanol ratio on unsaturated fatty acids yields In urea complexation, methanol was used as a solvent to dissolve the urea and fatty acids so that the urea and fatty acid could contact well and provide an appropriate crystalline environment. Since entrained methanol in the urea complexes was almost constant, when the methanol amount was increased, unsaturated fatty acids dissolved in the entrained methanol would be reduced, and the unsaturated fatty acids yield would be improved, as shown in Fig. 2. On the other hand, when the amount of methanol increased, more urea was dissolved, so it became more difficult to crystallize. However, as the solubility of urea in methanol was limited at low temperature, the unsaturated fatty acids’ content reduced slightly, as shown in Fig. 2. As amounts of methanol in excess of 5–6, the unsaturated fatty acids’ yields remain nearly constant because of good decentralization and less entrained unsaturated fatty acids. Considering the unsaturated fatty acid content and the filtrate yield, the methanol to fatty acids ratio 5 was selected as the optimal reaction condition.
The amount of urea added in the urea complexation process is an important parameter. Since urea is the primary crystalline substance, the amount of urea influences the amount of the complexation of saturated fatty acids. In the crystallization process, the urea crystal can form a hexagonal-hollow channel structure of 8.23 Å width (between the two sides), and can hold a saturated linear hydrocarbon chain with the cross-sectional size of 7.48 Å 4.99 Å (Smith, 1952), which facilitates the penetration of saturated fatty acids, that hence remain associated with the solid phase. However, the non-rotational double bonds in unsaturated fatty acids turn even the linear hydrocarbon chain into a cross-sectional size over 8.23 Å. This steric hindrance opposes unsaturated fatty acids0 penetration, leaving them in the methanol solution. The complexations were performed for varying FA-to-urea ratio, ranging from 1:0 to 1:3 (w/w) at temperature of 50 °C and stirring time 50 min. The mixture was cooled to a specific temperature of 20 °C. The
As the production process of methyl esters is mature, the reported method was used for methanolysis (Liu et al., 2014; Nie et al., 2006), and immobilized Candida sp. 99–125 was used as catalyst of WCO methanolysis. But for enzymatic isopropanolysis, there was no reported method, so the same method as methanolysis was tried. As shown in Fig. 1, from 0 h to 24 h, the isopropanolysis rate was quite fast. However, from 24 h to 40 h, the rate became slow, which indicated esterification and hydrolysis reaction rates reached a balance. At 40 h, the isopropyl esters yields
Fig. 1. Enzymatic hydrolysis process of waste cooking oil and isopropanolysis reaction course (Section 2.2 and 2.5). The fatty acids0 yields and isopropyl esters0 yields, respectively (4, h).
Fig. 2. The influence of methanol-to-FA ratio. The unsaturated fatty acids0 content and unsaturated fatty acids0 yields, respectively (j, d).
3.4. Enzyme catalyzed esterification of saturated fatty acids and isopropanol
M. Wang et al. / Bioresource Technology 174 (2014) 302–305
reached up to 98.9%. Therefore, 40 h was chosen as optimal isopropanolysis time.
3.5. The CFPP of the ester mixture Finally, the two esters, methyl unsaturated fatty acid esters and isopropyl saturated fatty acid esters, were mixed together. The CFPP (cold filter plugging point) of this mixture was measured, and the CFPP of this mixture decreased from 5 °C to 3 °C. This decrease was mainly contributed by the isopropyl saturated fatty acid ester. When the saturated fatty acids were turned to methyl esters, they had relatively high freezing point and made the biodiesel easy to crystallize (CFPP was 5 °C). However, once they were turned to isopropyl esters and had dendritic head structure, which differed from FA methyl esters, crystallization in the mixture became more difficult (CFPP was 3 °C). Therefore, the biodiesel which was composed of methyl and isopropyl esters had a lower CFPP than the whole methyl biodiesel.
4. Conclusions The new process of improving the low temperature performance of biodiesel had a significant effect. The CFPP (cold filter plugging point) of the esters mixture was decreased from 5 °C to 3 °C. This process was different from winterizing FAME. In this process, all the fatty acids are consumed, and the whole process did not have any material losses. Furthermore, using lipase, an environmentally friendly catalyst, greatly reduces the environmental pollution in comparison with the commonly used chemical catalysts.
Acknowledgements This research was financially supported by the Key Projects in the National Science & Technology Pillar Program during the 12th Five-year Plan Period (No. 2011BAD22B04), Amoy Industrial Biotechnology R&D and Pilot Conversion Platform (No. 3502Z20121009), the Fundamental Research Funds for the Central University (ZY1331), the Transformation of Agricultural Science and Technology Achievements Project (2013GB2C410539).
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