Energy Fuels 2010, 24, 2016–2019 Published on Web 02/24/2010
: DOI:10.1021/ef9011824
Conversion of Waste Cooking Oil to Biodiesel via Enzymatic Hydrolysis Followed by Chemical Esterification Md. Mahabubur Rahman Talukder,* Jin Chuan Wu, and Louisa Pei-Lyn Chua Institute of Chemical and Engineering Sciences, 1 Pesek Road, Jurong Island, Singapore 627833, Singapore Received October 18, 2009. Revised Manuscript Received December 23, 2009
Biodiesel (BD) is usually produced by alkali-catalyzed methanolysis of expensive edible oils. Although waste cooking oils (WCO) containing high free fatty acid (FFA) are inexpensive, they cannot be processed effectively by alkali catalysis because of the formation of soap, which reduces the BD yield and makes the downstream process complicated. A process consisting of enzymatic hydrolysis followed by chemical esterification was developed for the production of BD from WCO. The enzyme Candida rugosa lipase was used for the hydrolysis of WCO to fatty acid (FA). The complete conversion of WCO to FA was achieved after 10 h at a water to WCO ratio of 1:1 (v/v), 5 g of WCO, 5 mL of lipase-water solution (0.5 g/L), and temperature of 30 °C. After hydrolysis, FA was separated and converted to BD by chemical esterification using Amberlyst 15 (acidic styrene-divinylbenzene sulfonated ion-exchange resin) as a catalyst. The maximum BD yield of 99% was obtained after 2 h at a methanol to FA molar ratio of 4:1, 10 mL of FA-isooctane solution (0.35 M), 1 g of Amberlyst 15, and temperature of 60 °C. The activity of C. rugosa lipase slightly decreased with recycling, and FA yield after five cycles was 92%. Amberlyst 15 was repeatedly used for 100 cycles without loosing its activity. The developed two-step process has a potential to be used industrially as it can tolerate feedstocks containing a wide range of FFA and water.
acid catalysts can simultaneously catalyze esterification of FA and methanolysis of triglycerides.10,11 An acid-catalyzed reaction is slow and requires large stoichiometric excess of methanol, which leads the side reaction of methanol etherification.12 A two-step process, consisting of acid-catalyzed esterification followed by alkali-catalyzed methanolysis, is an alternative to convert feedstock with high FFA to BD.11 However, this twostep catalysis is not suitable for the feedstock like trap-grease in which water content may be as high as 55 wt %.13 Lipasecatalyzed methanolysis has attracted considerable attention for the production of BD as it requires mild reaction conditions and minimizes the separation and purification problems encountered in alkali and acid catalysis.14-17 Unfortunately, lipase is deactivated when more than one-third stoichiometric amount of methanol is present in the reaction.14-19 A catalystfree supercritical methanol method via hydrolysis followed by methyl esterification has been reported for the production of BD.20 The supercritical methanol process is very energy
Introduction Alkali-catalyzed methanolysis is usually adopted for the production of fatty acid methyl esters, which are collectively called biodiesel (BD).1-3 BD is mostly produced from expensive virgin vegetable oils, which accounts for 70-80% of its total production cost.4 Therefore, the need for processing cheaper feedstocks is of interest in BD research.5-9 Waste cooking oils (WCO) containing high free fatty acid (FFA) and water are inexpensive and could be a potential feedstock for BD industries. However, WCO cannot be processed effectively by an alkali process because FFA reacts with alkali catalysts, and the soap instead of BD is formed. The formation of soap reduces BD yield and causes the difficulties in downstream processing. The presence of water enhances the formation of soap as it causes the hydrolysis of triglycerides to fatty acid (FA). The aforementioned drawbacks of the alkali process have led researchers to seek catalytic and processing alternatives. Acid catalysts were used instead of alkali catalysts because
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*To whom correspondence should be addressed. Fax: þ65 63166182. E-mail:
[email protected]. (1) Lang, X.; Dalai, A. K.; Bakshi, N. N.; Reaney, M. J.; Hertz, P. B. Bioresour. Technol. 2001, 80, 53–62. (2) Meher, L. C.; Dharmagadda, V. S. S.; Naik, S. N. Bioreour. Technol. 2006, 97, 1392–1397. (3) Vicente, G.; Martinez, M.; Aracil, J. Bioresour. Technol. 2004, 92, 297–305. (4) Huang, C.; Zong, M. H.; Wu, H.; Liu, Q. P. Bioresour. Technol. 2009, 100, 4535–4538. (5) Halim, S. F. A.; Kamaruddin, A. H. Process Biochem. 2008, 43, 1436–1439. (6) Marchetti, J. M.; Miguel, V. U.; Errazu, A. F. Fuel 2007, 86, 906–910. (7) Talukder, M. M. R.; Wu, J. C.; Nguyen, T. B. V.; Ng, M. F.; Yeo, L. S. M. J. Mol. Catal. B: Enzym. 2009, 60, 106–112. (8) Talukder, M. M. R.; Wu, J. C.; Lau, S. K.; Cui, L. C.; Shimin, G.; Lim, A. Energy Fuels 2009, 23, 1–4. (9) Wang, Z. M.; Lee, J. S.; Park, J. Y.; Wu, C. Z.; Yuan, Z. H. Korean J. Chem. Eng. 2008, 25, 670–674. r 2010 American Chemical Society
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Energy Fuels 2010, 24, 2016–2019
: DOI:10.1021/ef9011824
Talukder et al.
consuming and requires a large excess of methanol (methanol-FA ratio 0.9:1, v/v) to suppress the backward reaction of BD to FA. In this study, the production of BD from WCO via C. rugosa lipase-catalyzed hydrolysis followed by Amberlyst 15-catalyzed esterification was investigated. The effect of different parameters on the hydrolysis of WCO and the esterification of FA were examined. The developed two-step process minimizes the aforementioned problems and has the advantage of feedstock flexibility over single-step alkali/acid or enzymatic transesterification: it can accept feedstock with any percentages of FFA and water. Experimental Section Materials. Amberlyst 15 and standard methyl esters (98 to 99%) were from Sigma. Candida rugosa lipase was from Meito Sangyo Co. Ltd., Osaka, Japan. WCO was collected from food courts in Singapore. The saponification value of WCO was 200-202 mg of KOH/g. The saponification value was determined by a method reported previously.21 The FFA and water content in WCO were 17.3 and 0.45 wt %, respectively. HPLC grade methanol, isopropanol, isooctane, and hexane were from J.T. Baker, USA. All chemicals, unless mentioned otherwise, were of analytical grade and used as received. Hydrolysis of WCO. Five grams of WCO was preheated in a glass bottle (80 mL size) at 30 °C and 250 rpm for 30 min by a shaker incubator. The reaction was initiated by adding an appropriate amount of lipase solution. This lipase solution was prepared by dissolving C. rugosa lipase in deionized water (pH 6.8-7.4) and was directly used. The lipase concentration was varied according to 0.01-0.1 wt % of WCO. The water to WCO ratio was varied in the range of 0.1:1 to 3:1 (v/v). Separation of FA after Hydrolysis. Isooctane (50 mL) was added to the reaction mixture and mixed at 30 °C and 250 rpm for 15 min. The mixture was centrifuged at 4000 rpm and 25 °C for 10 min. The upper layer containing FA in isooctane was separated and used as feedstock for Amberlyst 15-catalyzed methyl esterification. To measure the FA concentration, 5 mL of upper layer was mixed with 20 mL of ethanol-acetone (50/50, v/v) solution and titrated against 0.2 N NaOH using phenolphthalein as an indicator. The molar FA yield is calculated as total FA (initial plus formed) divided by theoretical value. Esterification of FA in Isooctane. Ten milliliters of FAisooctane solution (0.35-0.36 M) and an appropriate amount of methanol were mixed in a glass bottle (80 mL size) at 60 °C for about 15 min by a shaker incubator. The methanol to FA molar ratio was varied from 1:1 to 4:1. The reaction was initiated by adding Amberlyst 15. The Amberlyst 15 amount was varied from 0.2 to 0.4 g. Determination of Water Content. The water content in WCO is determined by the Karl Fischer titration method using a Karl Fischer moisture titrator (Mettler-Toledo, Greifensee, Switzerland). A sample weighed exactly was titrated using HYDRANAL-composite 5 (Sigma-Aldrich Laborchemkalien, Seelze, Germany) as the Karl Fisher reagent. High Performance Liquid Chromatography (HPLC) Analysis of BD. After the specified time of methyl esterification in isooctane, the whole sample was centrifuged at 4000 rpm and 25 °C for 15 min, and BD in the upper layer was analyzed. The method used for HPLC analysis of BD was previously reported.22,23 HPLC (Waters 2695, USA) was equipped with a UV detector
Figure 1. Effect of water to WCO ratio (v/v) on specific activity of C. rugosa lipase. Reaction conditions: 5 g of WCO; lipase concentration in water, 1 g/L; temperature, 30 °C; time, 20 min; and shaking speed, 250 rpm.
(Waters 2487, USA) and a prevail-C18 5u column (4.6 250 mm, Altech Inc., USA). The UV wavelength and the column temperature were set at 210 nm and 40 °C, respectively. The mobile phase consisted of three different components: hexane, isopropanol, and methanol. Reservoir A contained methanol, and reservoir B contained a mixture of isopropanol and hexane (5:4, v/v). The gradient went from 100% A to 50% A þ 50% B linearly over 30 min. The flow rate of the mobile phase was 1 mL/min, and the sample injection volume was 10 μL. BD was quantified according to the external calibration curves. Methyl oleate, methyl palmitate, methyl stearate, methyl linoleate, and methyl myristate were used as standards for BD. BD yield was calculated as the amount of BD produced (actual value in grams) divided by the theoretical value in grams. The average molecular weight of FA from WCO was 275 g/mol.
Results and Discussion C. rugosa Lipase-Catalyzed Hydrolysis of WCO. Among the lipases from various sources, C. rugosa lipase has been reported as one of the most active and versatile enzymes.24-26 Hence, this lipase was chosen for hydrolysis of WCO to FA. Lipase is a surface active enzyme and binds with substrates at the oil-water interface.27,28 Since interfacial area depends on water to oil ratio (v/v),29,30 the lipase specific activity (moles of FA produced per hour per gram of lipase) at different water to WCO ratios was investigated. The experiment was performed by varying the amount of water while keeping the lipase concentration in water (1 g/L) and the amount of WCO (5 g) constant. Figure 1 shows that the specific activity of lipase reached maximum at a water to oil ratio of 0.5:1, after which it dropped. This result indicates that the interfacial area could reach a maximum level at a water to oil ratio of 0.5:1 (v/v). Al-Zuhair et al.31 have studied the effect of different (24) Benjamin, S.; Pandey, A. Yeast 1998, 14, 1069–1087. (25) Macrae, A. R.; Hammond, R. C. Genetic Eng. Rev. 1985, 3, 193– 217. (26) Maria, P. D.; Montero, J. M. S.; Sinisterra, J. V.; Alcantara, A. R. Biotechnol. Adv. 2006, 24, 180–196. (27) Benjamin, S.; Pandey, A. Bioresour. Technol. 1996, 55, 167–170. (28) Mingarro, I.; Novarro, H.; Braco, L. Biochem. 1996, 35, 9935– 9944. (29) Kim, M. K.; Rhee, J. S. Enzyme Microb. Technol. 1993, 15, 612– 616. (30) Mukataka, S.; Kobayashi, T.; Sato, S.; Takahashi, J. J. Ferment. Technol. 1987, 65, 23–29. (31) Al-Zuhair, S.; Ramachandran, K. B.; Hasan, M. J. Chem. Technol. Biotechnol. 2004, 79, 706–710.
(21) Mendham, J.; Denney, R. C.; Barnes, J. D.; Thomas, M. Vogel’s Textbook of Quantative Chemical Analysis; Pearson Education: Harlow, England, 2000; pp 364. (22) Holcapek, M.; Jandera, P.; Fischer, J.; Prokes, B. J. Chromatogr., A 1999, 858, 13–31. (23) Chen, J. W.; Wu, W. T. J. Biosci. Bioeng. 2003, 95, 466–469.
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Energy Fuels 2010, 24, 2016–2019
: DOI:10.1021/ef9011824
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Figure 2. Time courses hydrolysis of WCO at different water to WCO ratios. Reaction conditions: 5 g of WCO; lipase 0.05 wt % of WCO; temperature, 30 °C; and shaking speed, 250 rpm.
Figure 3. Recycling of C. rugosa lipase-catalyzed hydrolysis of WCO. Reaction conditions: 2 g of WCO; 2 mL of lipase-water solution (0.25 g/L); temperature, 30 °C; time, 24 h; and shaking speed, 250 rpm.
parameters on the specific interfacial area of the palm oilwater mixture, which is very similar to the present work particularly at a water to oil ratio of >1:1 at which the mixture can be considered as oil-in-water emulsion. They have found that the interfacial area per unit volume increased with the increase in volume fraction of dispersed phase (palm oil) up to 0.5. Although the volume fraction of dispersed phase (WCO) in the present work dropped at a higher water to oil ratio, this may not alter the total interfacial area as the total volume of the mixture also increased. However, the mixing efficiency could be reduced due to the increase in total volume of the mixture, thereby increasing the droplet size. Zhou et al.32,33 have proposed an empirical model that correlates the Sauter mean drop diameter with the maximum kinetic energy dissipation (εmax), agitation speed (N), and impeller diameter (D). They observed that Sauter mean drop diameter increased with the decrease in εmax, N and D. This suggests that the total interfacial area at a higher water to oil ratio decreased due to the larger droplet size. Hence, the specific activity of lipase at a water to oil ratio of >1:1 decreased. Lipase-catalyzed hydrolysis of oil is an equilibrium reaction, and the higher water content shifts the reaction equilibrium to the right. From this viewpoint, the time course hydrolysis of WCO at a relatively higher water to oil ratio of 1:1 (v/v) was compared with that at a ratio of 0.5:1 (v/v) (Figure 2). The lipase amount was kept constant at 0.05 wt % of WCO. The hydrolysis of WCO at a water to oil ratio of 0.5:1 reached the equilibrium FA yield (93%) after 10 h. FA yield was improved to almost 100% by the increase in water to oil ratio from 0.5:1 to 1:1. This result suggests that the reverse hydrolysis cannot occur at a higher water to oil ratio leading to the complete conversion of WCO to FA. A water to oil ratio of 1:1 was chosen for the subsequent experiments. Recycling of C. rugosa Lipase. Although the rapid development of molecular biology techniques help to reduce the production cost of lipase, lipase recycling is still essential in the production of low value product like BD. For investigating the recyclability of C. rugosa lipase, the hydrolysis of WCO was performed under the following conditions: 2 g of WCO; 2 mL of lipase-water solution (0.25 g/L); temperature, 30 °C; time, 24 h; and shaking speed, 250 rpm. After each cycle of reaction, the sample was mixed with 20 mL of
isooctane at 30 °C and 250 rpm for 15 min. The mixture was centrifuged at 4000 rpm and 25 °C for 10 min. The aqueous phase containing lipase and glycerol was separated from the organic phase (FA and isooctane) and subsequently used without further treatment. Figure 3 shows that C. rugosa lipase was quite stable: FA yield over 5 cycles is 92%. It has been reported that lipase can be stabilized by maintaining the glycerol concentration in the aqueous phase within the range of 10 to 40% by weight throughout the hydrolysis of oils or fats.34 Therefore, the byproduct glycerol might have a positive effect on the recycling of C. rugosa lipase. The slight decrease in FA yield was attributed to the accumulation of glycerol in the aqueous phase, which facilitated the reverse hydrolysis and increased the viscosity. Amberlyst 15-Catalyzed Methyl Esterification of FA. Amberlyst 15 is an efficient, reusable, and commercially available catalyst. Compared with other solid acid catalysts, Amberlyst 15 is relatively cheaper, well studied, and used in many industries. Hence, this catalyst was chosen for esterification of FA. The time courses of methyl esterification of FA in isooctane at different methanol concentrations are shown in Figure 4. The reaction with a stoichiometric amount of methanol (i.e., methanol to FA molar ratio 1:1) reached the equilibrium BD yield (50%) after 5 h. With an increase in methanol concentration from 1:1 to 2:1 molar ratio, the reaction equilibrium shifted to the right giving a higher BD yield of 99% after 4 h. With further increase in methanol concentration to a 4:1 molar ratio, the maximum BD yield remained the same. However, the time required for reaching the equilibrium became shorter (2 h). The time courses of methyl esterification at different amounts of Amberlyst 15 are shown in Figure 5. As the Amberlyst 15 amount was increased, there was a sudden surge in the formation of BD, followed by a slower rate of increase. The increase in reaction rate was due to the increase in total number of Amberlyst 15 active sites available for the reaction. The reaction rate reached an upper limit when Amberlyst 15 loading exceeded 2 g. It is evident from Figure 5 that 1 g of Amberlyst 15 (100 wt % of FA) is sufficient to achieve a BD yield of 99% after 2 h. Recycling of Amberlyst 15. One of the most important advantages of heterogeneous catalyst is its easy recovery and (34) Masanobu, T.; Hidetoshi, W.; Masaru, S. U.S. Patent 5032515, 1991.
(32) Zhou, G.; Kresta, S. M. Chem. Eng. Sci. 1998, 58, 2063–2079. (33) Zhou, G.; Kresta, S. M. AIChE J. 1996, 42, 2476–2490.
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Energy Fuels 2010, 24, 2016–2019
: DOI:10.1021/ef9011824
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Figure 6. Recycling of Amberlyst 15. Reaction conditions: 10 mL of FA-isooctane solution (0.35-0.36 M); methanol to FA molar ratio, 4:1; temperature, 60 °C; time, 2 h; and shaking speed, 250 rpm.
without losing its activity. On the contrary, Amberlyst 15 without washing and freeze-drying loses the activity with recycling, and BD yield after six cycles was only 35%. Water formed during the esterification was adsorbed onto Amberlyst 15 and inhibited the reaction. Hence, BD yield with recycled Amberlyst 15 decreased. To understand whether both methanol washing and freeze-drying are necessary to maintain a high BD yield, their effects on the recycling of Amberlyst 15 were investigated. BD yield with methanol washing alone was 70% at the second cycle after which it remained almost the same over the cycles tested (data not shown). Methanol washing alone was insufficient to completely remove the water adsorbed onto Amberlyst 15, and the remaining water shifted the reaction equilibrium to the left, giving lower BD yield. Freeze-drying alone could maintain BD yield at a level of 97 to 98% over 10 cycles. These results suggest that the removal of water adsorbed onto Amberlyst 15 was the most crucial factor to recycle Amberlyst 15 and the methanol washing prior to freeze-drying may not be necessary.
Figure 4. Time course esterification of FA at different methanol concentrations. Reaction conditions: 10 mL of FA-isooctane solution (0.35-0.36 M); 1 g of Amberlyst 15; temperature, 60 °C; and shaking speed, 250 rpm.
Conclusions A two-step process consisting of C. rugosa lipase-catalyzed hydrolysis followed by Amberlyst 15-catalyzed esterification was employed for the production of BD from WCO containing high FFA (17.5 wt %) and water (0.45 wt %). The water to WCO ratio (v/v) significantly influenced the activity of C. rugosa lipase and the equilibrium FA yield. Methanol concentration and Amberlyst 15 amount play an important role in efficient conversion of FA to BD. The removal of water adsorbed onto Amberlyst 15 is essential to recycle Amberlyst 15. A key attribute of the developed process is its ability to utilize a wide variety of feedstocks such as vegetable oils, animal fats, and waste greases.
Figure 5. Effect of Amberlyst 15 loading on esterification of FA. Reaction conditions: 10 mL of FA-isooctane solution (0.35-0.36 M); methanol to FA molar ratio, 4:1; temperature, 60 °C; and shaking speed, 250 rpm.
repeated use over an extended period of time. Experiments were performed to investigate the recyclability of Amberlyst 15. After each cycle (2 h), Amberlyst 15 was filtered using filter paper (Whatman 125), washed with 10 mL of methanol, freeze-dried for 12-15 h, and subsequently reused. The reaction was carried out at 60 °C with 1 g of Amberlyst 15, 10 mL of FA-isooctane solution (0.35-0.36 M), and 0.6 mL of methanol (methanol to FA molar ratio, 4:1). Figure 6 shows that when Amberlyst 15 was washed with methanol and freeze-dried, it could be repeatedly used over 100 cycles
Acknowledgment. Financial support from the Agency for Science Technology and Research (A*STAR) of Singapore is gratefully acknowledged.
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