Journal of Oleo Science Copyright ©2015 by Japan Oil Chemists’ Society doi : 10.5650/jos.ess14103 J. Oleo Sci. 64, (1) 69-74 (2015)
Enzymatic Production of Biodiesel from Microalgal Oil using Ethyl Acetate as an Acyl Acceptor Razieh Shafiee Alavijeh1, Fatemeh Tabandeh2＊ , Omid Tavakoli1＊ , Aliasghar Karkhane2 and Parvin Shariati2 1 2
School of Chemical Engineering, College of Engineering, University of Tehran, 16 Azar Street, Tehran, Iran. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract: Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃ . The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction. Key words: biodiesel, microalgae oil, enzymatic transesterification, Novozym 435, ethyl acetate, acyl acceptor 1 Introduction Biodiesel, as a non-petroleum-based diesel fuel, is usually produced by the transesterification of a variety of oils including vegetable oils, animal fats, waste cooking oil and algal oil1）. It is generally regarded as clean and environmentally safe, and thus considered a technically and economically competitive fuel when compared to other conventional fossil fuels, such as diesel2）. Biodiesel production from microalgal oil is more promising than other types of oil feedstock microalgae have a fast rate of growth, can be grown in almost all aquatic ecosystems and provide a wide range of different fatty acids which can be used for food, feed and biodiesel applications1, 2）. The other advantages of biodiesel production by microalgae are: i. they can be easily cultivated in seawater, ii. microalgae do not occupied arable lands, so they do not compete with agricultural plants which used as food, iii. they produce high yields of oil per unit area in comparison to oil crops1, 2）. Transesterification is a common method used to convert triglycerides （TG）into biodiesel. This process involves exchanging the organic group of an ester（e.g. TG）with the organic group of an acyl acceptor3, 4）. Carboxylic acids, alcohols, or other esters can be used as acyl acceptors. However, when
alcohol is used as the acyl acceptor, an impure by-product, glycerol（also known as glycerin）, is produced during the transesterification process5）. The glycerin fraction of the transesterification reaction, regarded as spent liquor, can be harmful to the environment6）. An effective method to overcome the glycerin surplus is the transesterification of TG with esters such as methyl or ethyl acetate, producing triacylglycerol instead of glycerol. The mixture of triacylglycerol and fatty acid methyl/ethyl esters can be used directly as biodiesel7）. Biodiesel can be successfully produced using different catalytic processes involving chemical or enzyme catalysts 8）. Enzymatic reaction is of much interest as it is an environmentally friendly process, which produces high purity products9）. Various kinds of acyl acceptors consisting of alcohols and esters can be employed in transesterification reactions catalyzed by lipolytic enzymes. Methanol and ethanol have been the most frequently-used alcohols for the industrial production of biodiesel because of their price and availability. But such types of alcohols have been reported to have a stronger denaturing action on lipase than long-chain aliphatic alcohols10）. The degree of inactivation seems to be inversely proportional to the number of
＊
Correspondence to: F. Tabandeh, Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Omid Tavakoli, School of Chemical Engineering, College of Engineering, University of Tehran, 16 Azar Street, Tehran, Iran. E-mail: [email protected], [email protected] Accepted September 1, 2014 (received for review May 26, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs
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R. S. Alavijeh, F. Tabandeh, O. Tavakoli et al.
carbon atoms of the alcohol used in the transesterification reaction11）. The most common strategy to diminish the risk of enzyme deactivation is stepwise addition of alcohol12−14）. The use of different acyl acceptors such as methyl/ethyl acetate is an alternative and effective way to overcome this problem, because a short-chain alcohol is substituted with the short-chain alkyl acetate, which has no adverse effects on enzyme activity15）. Various acyl acceptors have been employed for transesterification of vegetable oils to biodiesel16−18）. Xu et al.16） suggested using methyl acetate for biodiesel production from renewable oils in a solvent-free medium. In fact, the production of ethyl esters is preferred to methyl esters because ethyl esters have lower pour and cloud points and higher flash and combustion points, so they increase heat content and the cetane number of biodiesel18, 19）. The present study focuses on the main process variables affecting the enzymatic transesterification reaction in the presence of ethyl acetate as an acyl acceptor for the production of biodiesel from microalgal oil as a suitable feedstock. The effect of ethyl acetate on the stability of commercial immobilized lipase, Novozym 435, was also investigated. Crude microalgal oil was employed in this study because the oil purification processes impose extra expenses and increase the final cost of the product.
2 Materials and methods 2.1 Materials Candida antarcitica lipase B（Novozym 435）, immobilized on macroporous acrylic resin with a known activity of approximately 10,000 PLU/g （propyl laurate units/g） , was a gift sample from Novo Nordisk（ A.S., Denmark-Tehran Office） . The powdered form of Chlorella vulgaris was obtained from Qeshm Sina Microalgae Company （Iran） . Standard fatty acid esters were used as reference and purchased from Sigma Aldrich（USA）. All other reagents and solvents used were of analytical grade. 2.2 Oil extraction First, 10 g of microalgal powder was mixed with 150 ml of distillated water. The mixture was sonicated at 70 amplitude for 15 min （Sonicator, Hielscher, Germany） in order to disrupt the microalgae cell wall. Then, a bi-phase solvent solution of chloroform-methanol（1:1 v/v）was added and the mixture was vigorously shaked for 5 min. The resulting mixture was centrifuged at 6708×g for 10 min. The upper polar phase was discarded and the lower phase containing the chloroform and microalgal lipid was separated for further use. Chloroform was evaporated in a hood under ambient temperature20）. The yield of extraction from microalgal oil was 12％.
2.3 Transesterification reaction Novozym 435 was added to mixtures of crude microalgal oil（0.25 g）and ethyl acetate, at different molar ratios of 12:1, 13:1, 14:1, 15:1, 20:1, 25:1（ethyl acetate to oil）. The molecular weight of oil was calculated based on its fatty acid content, obtained by gas chromatography. Different amounts of enzyme（30, 40, 50, 60％ w/w of oil）were then added, and the resulting mixtures containing the reactants were placed in a shaker incubator at different temperatures （30, 35, 40, 45℃） , with shaking rate of 200 rpm for 48 h or 72 h. At the end of the reaction, the enzyme was separated by filtration, the unreacted ethyl acetate was distilled into a concentrator（ Eppendorf Concentrator 5301, Germany）and the crude product was analyzed by gas chromatography. Lipase stability was studied by measurement of the fatty acid ethyl ester（FAEE） content following the transesterification reaction. The mean differences in various groups of the obtained data were compared by one-way ANOVA. Pvalues less than 0.05 were considered significant. Statistical calculations were carried out with the Minitab software version 16. 2.4 Analytical methods The fatty acid content of microalgal oil was measured by gas chromatography（GC）21）. The methyl ester present in the reaction mixture was analyzed using a GC-3800CP gas chromatography system（Varian Crop., Netherlands）connected to a cpsil-5CB capillary column（0.32 mm×30 m, Varian, Netherland）. Methyl heptadecanoate was used as an internal standard, 5 mg of which was mixed with 50 mg of crude product and 1 ml of hexane. Subsequently, 1 μl of the resulting solution was injected into the GC. The column temperature was held at 150℃ for 1 min and then subsequently raised to 200℃ at the rate of 20℃ /min, to 207℃ at the rate of 1℃ /min, to 300℃ at a rate of 30℃ /min and finally maintained at this temperature for 24 min. The temperatures for the injector and flame ionization detector （FID）were set at 270 and 300℃, respectively. The fatty acid ethyl ester（FAEE） content, expressed as a mass fraction in percent, is calculated using the following formula: Σ （A−AIS） CIS×VIS ×100 Eq.1 × FAEE％＝ M AIS Where, ΣA is the total peak area from the fatty acid ethyl esters C14:0 to C24:1, AIS is the peak area of the internal standard（methyl heptadecanoate）, CIS is the concentration of the internal standard solution in mg/mL, V IS is the volume of the methyl heptadecanoate solution in mL and M is the mass of the sample in mg. In order to quantify the FAEE content and identify the by-products of the reaction, the crude transesterification product（1.0 g）was dissolved in ethyl acetate-hexane（1:10 v/v）and then loaded on a column（22 cm×1.5 cm）packed with silica gel（15 g, 60 mesh size）. Elution of the ethyl
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ester fraction was accomplished with an ethyl acetate-hexane mixture. The last collected fraction contained the byproduct formed during transesterification. This fraction was then subjected to thin layer chromatography（TLC）to separate and identify its constituents containing FAEE and triacetin 18）.
3 Results and discussion 3.1 Effect of the ethyl acetate to oil molar ratio on FAEE yield Different molar ratios of ethyl acetate to oil, ranging from 12:1 to 25:1, were used. The fatty acid compositions of obtained esters were C16:0 palmitic acid, C18:0 stearic acid, C18:1 oleic acid, C18:2 linoleic acid and C18:3 linolenic acid. The highest amount of FAEE was obtained at an ethyl acetate to oil molar ratio of 13:1 when an enzyme dosage of 40％ （w/w enzyme/oil） was used at 40℃ for 72 h （Fig. 1）. The reaction medium was insoluble and highly viscous at the 12:1 molar ratio. However, no significant difference was observed when the molar ratio of ethyl acetate to oil was increased to 17:1 （p＜0.05） . The FAEE yield was reduced at higher molar ratios because of the dilution of the reaction medium. In other reports, optimum ratios of ethyl acetate to oil were 12:1 and 11:1, respectively, when various vegetable oils were used as the substrate17, 18）. In the present study, crude microalgal oil contained different kinds of complex compounds that include polar lipids wax esters, sterols and hydrocarbons such as tocopherols, carotenoids and terpenes and phytylated pyrrole derivatives such as chlorophylls22）. Therefore, this can be considered as a -viscous substrate, and it seems that higher amounts of ethyl acetate are necessary to improve the transesterification reaction. 3.2 Effect of enzyme dosage on FAEE yield Maximum FAEE percentage yields of 46.16±4.72 and 47.21±2.23 were obtained at a molar ratio of 13:1 and enzyme doses of 50％ and 40％, respectively（Fig. 2）. An optimum enzyme dosage of 10％ has previously been reported in achieving a 92.7％ FAEE yield from the transesterification of sunflower oil by Novozym 43518）. This is much lower than that used for transesterification of microalgal oil in this study, which may be due to the degree of
Fig. 1 Effect of different ethyl acetate to oil molar ratios on FAEE yield during transesterification of microalgal oil catalyzed by Novozym 435 at a dose of 50%, and incubated at 40℃ with shaking rate of 200 rpm for (■) 48 h and (□) 72 h.
Fig. 2 Effect of enzyme dosage on FAEE yield during transesterification of microalgal oil at 40℃ and 200 rpm for 48 h. ( ) 30% enzyme dosage, ( ) 40% enzyme dosage, ( ) 50% enzyme dosage and ( ) 60% enzyme dosage. purity of this crude oil interfering with the enzyme activity. Moreover, the analysis of the fatty acid content of microalgal oil showed that the amounts of saturated fatty acids （C16:0 and C18:0）were more than those in vegetable oils such as sunflower and Jatropha oils（Table 1）. The main fatty acid components of Chlorella vulgaris were found as palmitic acid（40.92％）, stearic acid（2.41％）, oleic acid （21.20％）, linoleic acid（33％）and linolenic acid（2.28％）. Hence, another reason for requiring a higher enzyme
Table 1 Fatty acid composition of methyl esters prepared from microalgal oil. Type of oil
Fatty acid composition (% w/w)
Ref
16:0
16:1
18:0
18:1
18:2
18:3
20:0
20:1
22:0
24:0
Microalgal oil
40.92
2.41
21.20
33.17
2.28
－
－
－
－
－
This study
Sunflower oil
5.70
－
46.50
44.80
－
－
－
－
－
－
18
Jatropha oil
16.40
1.00
6.20
37.00
39.20
－
0.20
－
－
－
18
71
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dosage may be due to the hydrolyzing of these saturated fatty acids. 3.3 Effect of temperature on FAEE yield The transesterification reaction was carried out at a temperature range of 30℃ to 50℃. As shown in Fig. 3, the FAEE yield increased when temperature increased from 30℃ to 40℃, and decreased when a temperature of 50℃ was used. A significant difference was observed at 40℃ when compared to other experiments（p＜0.05）. Accordingly, 40℃ was selected as the optimum temperature. The optimum temperatures of 50℃ and 40℃ have also been previously reported to achieve FAEE yields of 92.7％ and 91.0％, respectively from the transesterification of sunflower oil and soybean oil in the presence of ethyl acetate and methyl acetate when using Novozym 43518, 23）. 3.4 Time profile of FAEE production The reactants were exposed to optimum thermal,
enzyme dosage and molar ratio conditions and were then sampled at different time intervals. The FAEE yield was found to increase during a time period of 72 h（Fig. 4）, showing a significant difference at 72 h when compared to the preceding intervals（p＜0.05）. Similar studies using ethyl/methyl acetate as acyl acceptors for the transesterification of vegetable oils have also been carried out, however, optimum reaction times much lower than that of the transesterification of microalgal oil of this study have been reported17, 18）. This again may be due to the use of the enzyme dosage as mentioned above, which suggests that the enzyme in the crude and impure media needs a greater span of time to catalyze the reaction. 3.5 Stability of immobilized lipase in the presence of ethyl acetate The effect of ethyl acetate on the stability of Novozym 435 was determined at repeated transesterification cycles. After each cycle, the enzyme sample was removed and washed with cold acetone. It was then applied to the next cycle using fresh substrate. The transesterification reaction was carried out in 7 cycles sequentially. For comparative purpose, the ethanolysis reaction of microalgal oil by Novozym 435（50％）at 40℃ and 200 rpm involving a threestep addition of ethanol was also carried out in repeated cycles. The results showed that the relative yield of FAEE was approximately 100％ in the presence of the acyl acceptor; ethyl acetate, after 7 cycles, while it decreased dramatically and reached zero after 3 cycles in the presence of ethanol（Fig. 5）. This indicated that ethyl acetate has no
Fig 3 Effect of temperature on ethyl ester yield during interestrification of microalgal oil using ethyl acetate to oil molar ratio of 13:1 at 200 rpm for 48 h.
Fig 4 Time profile of FAEE production during transesterification of microalgal oil catalyzed by Novozym 435 (50% based on oil weight) at 40℃ and 200 rpm, using an ethyl acetate to oil molar ratio of 13:1.
Fig 5 Operational stability of lipase during the transesterification reaction of microalgal oil at 40℃ and 200 rpm using 50% Novozym 435. ( □) Transestrification by ethyl acetate, the molar ratio of ethyl acetate to oil was 13:1 and duration of the transesterification reaction was 72 h. (■) Ethanolysis, for the ethanolysis reaction, the molar ratio of ethanol to oil was 3:1 (three-step addition) during 48 h of the reaction process.
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adverse effects on the enzyme and can be used in largescale for further studies. Modi et al.18） have also studied the stability of Novozym 435 in the presence of ethyl acetate and sunflower oil as substrates. They have shown that lipase is stable in all ten cycles during the transesterification reaction.
4 Conclusion One of the challenges in the commercial production of biodiesel by enzymatic transesterification is the high cost of enzymes, which resulted to elevate the price of the final biodiesel product. Therefore, maintenance of lipase activity during the production cycles is essential. In addition, using a variety of vegetable oils as fuel and energy sources, ultimately leads to interactions with the food chain. Hence, microalgal oil has been suggested as an alternative feedstock for biodiesel production. Microalgae can be grown under different conditions and do not require fertile land and rainy weather. Furthermore, they store high levels of lipid, thus making them appropriate sources of fuel. The results of this study have established ethyl acetate as an acyl acceptor and microalgae as a source of triglycerides for the biodiesel production process. In fact, ethyl acetate has no negative impact on the operational lipase activity during the transesterification reaction, and also produces the valuable by-product, triacetin （glycerol triacetate）, that has widespread applications in the food, printing, tanning, cigarette, cosmetic, pesticide and pharmaceutical industries. Triacetin can also be used as a fuel additive, in the form of an antiknock agent, which can reduce knocking in gasoline engines and improve cold flow and viscosity properties of biodiesel. Also, under the optimized conditions of process variables, and with higher enzyme activities（higher than Novozym 435）, the enzymatic production of biodiesel becomes competitive with the conventional processes that involve acid and alkaline catalytic reactions.
Acknowledgements The work described in this paper was supported by NIGEB（Grant No. 429）and Iran National Science Foundation（INSF Grant No. 89001407）, and would be appreciated.
References 1）Taher, H.; Al-Zuhair, S.; AlMarzouqui, A.; Hashim,I. Extracted fat from lamb meat by supercritical CO2 as feedstock for biodiesel production. Biochem. Eng. J.
55, 23-31（2011）. 2）Demirbas, A. Progress and recent trends in biodiesel fuels. Energy Convers. Manage. 50, 14-34（2009）. 3）Fan, X.; Burton, R. Recent development of biodiesel feedstocks and the applications of glycerol: a review. Open Fuels Energy Sci. J. 2, 100-109（2009）. 4）Helwani, Z.; Othman, M.; Aziz, N.; Fernando,W.; Kim, J. Technologies for production of biodiesel focusing on green catalytic techniques: a review. Fuel Process. Technol. 90, 1502-1514（2009）. 5）Ranganathan, S. V.; Narasimhan, S. L.; Muthukumar, K. An overview of enzymatic production of biodiesel. Bioresour. Technol. 99, 3975-3981（2008）. 6）Xu Y.; Liu, H.; Du, W.;Sun, Y.; Ou, X.; Liu, D. Integrated production for biodiesel and 1, 3-propanediol with lipase-catalyzed transesterification and fermentation. Biotechnol. Lett. 31, 1335-1341（2009）. 7）Dubé, M.; Tremblay, A.; Liu, J. Biodiesel production using a membrane reactor. Bioresour. Technol. 98, 639-647（2007）. 8）Basr, i M.; Heng, A.; Razak, C.; Yunus, W. W.; Ahmad, M.; Rahman, R.; Ampon, K.; Salleh, A. Alcoholysis of palm oil mid-fraction by lipase from Rhizopus rhizopodiformis. J. Am. Oil Chem. Soc. 74, 113-116（1997）. 9）Miao, X.; Wu, Q. Biodiesel production from heterotrophic microalgal oil. Bioresour. Technol. 97, 841-846 （2006）. 10）Nelson, L. A., Foglia, T. A.; Marmer, W. N. Lipase-catalyzed production of biodiesel. J. Am. Oil Chem. Soc. 73, 1191-1195（1996）. 11）Chen, J.-W.; Wu, W.-T. Regeneration of immobilized Candida antarctica lipase for transesterification. J. Biosci. Bioeng. 95, 466-469（2003）. 12）Soumanou, M. M.; Bornscheuer U. T. Improvement in lipase-catalyzed synthesis of fatty acid methyl esters from sunflower oil. Enzyme Microb. Technol. 33, 97103（2003）. 13）Chen, G.; Ying, M.; Li, W. Enzymatic conversion of waste cooking oils into alternative fuel-biodiesel. Appl. Biochem. Biotechnol. 132, 911-921（2006）. 14）Lu, J.; Nie, K.; Xie, F.; Wang, f.; Tan, T.; Enzymatic synthesis of fatty acid methyl esters from lard with immobilized Candida sp. 99-125. Process Biochem. 42, 1367-1370（2007）. 15）Su, E.-Z.; Xu W.-Q.; Gao, K.-L.; Zheng, Y.; Wei, D.-Z. Lipase-catalyzed in situ reactive extraction of oilseeds with short-chained alkyl acetates for fatty acid esters production. J. Mol. Catal. B: Enzym. 48, 28-32 （2007）. 16）Xu, Y.; Du, W.; Liu, D.; Zeng, J. A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium. Biotechnol. Lett. 25, 1239-1241 （2003）. 17）Du, W.; Xu, Y.; Liu, D.; Zeng, J. Comparative study on 73
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lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors. J. Mol. Catal. B: Enzym. 30, 125-129 （2004） . 18）Modi, M. K.; Reddy, J.; Rao, B.; Prasad, R. Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor. Bioresour. Technol. 98, 1260-1264 （2007） . 19）Encinar, J.; Gonzalez, J.; Rodriguez, J.; Tejedor, A. Biodiesel Fuels from Vegetable Oils: Transesterification of Cynara c ardunculus L. Oils with Ethanol. Energy Fuels. 16, 443-450 （2002） . 20）Tran, D.-T.; Yeh, K.-L.; Chen, C.-L.; Chang, J.-S. Enzymatic transesterification of microalgal oil from Chlo-
rella vulgaris ESP-31 for biodiesel synthesis using immobilized Burkholderia lipase. Bioresour. Technol. 108, 119-127（2012）. 21）Phan, A. N.; Phan, T. M. Biodiesel production from waste cooking oils. Fuel 87, 3490-3496（2008）. 22）Bisen, P. S.; Sanodiya, B. S.; Thakur, G. S.; Baghel, R. K.; Prasad, G. Biodiesel production with special emphasis on lipase-catalyzed transesterification. Biotechnol. Lett. 32, 1019-1030（2010）. 23）Huang, Y.; Yan, Y.; Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor. Zeitschrift für Naturforschung. C, J. Biosciences. 63, 297-302 （2008）.
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Enzymatic Production of Biodiesel from Microalgal Oil using Ethyl Acetate as an Acyl Acceptor Razieh Shafiee Alavijeh1, Fatemeh Tabandeh2＊ , Omid Tavakoli1＊ , Aliasghar Karkhane2 and Parvin Shariati2 1 2
School of Chemical Engineering, College of Engineering, University of Tehran, 16 Azar Street, Tehran, Iran. Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran
Abstract: Microalgae have become an important source of biomass for biodiesel production. In enzymatic transesterification reaction, the enzyme activity is decreased in presence of alcohols. The use of different acyl acceptors such as methyl/ethyl acetate is suggested as an alternative and effective way to overcome this problem. In this study, ethyl acetate was used for the first time in the enzymatic production of biodiesel by using microalga, Chlorella vulgaris, as a triglyceride source. Enzymatic conversion of such fatty acids to biodiesel was catalyzed by Novozym 435 as an efficient immobilized lipase which is extensively used in biodiesel production. The best conversion yield of 66.71% was obtained at the ethyl acetate to oil molar ratio of 13:1 and Novozym 435 concentration of 40%, based on the amount of oil, and a time period of 72 h at 40℃ . The results showed that ethyl acetate have no adverse effect on lipase activity and the biodiesel amount was not decreased even after seven transesterification cycles, so ethyl acetate has a great potential to be substituted for short-chain alcohols in transesterification reaction. Key words: biodiesel, microalgae oil, enzymatic transesterification, Novozym 435, ethyl acetate, acyl acceptor 1 Introduction Biodiesel, as a non-petroleum-based diesel fuel, is usually produced by the transesterification of a variety of oils including vegetable oils, animal fats, waste cooking oil and algal oil1）. It is generally regarded as clean and environmentally safe, and thus considered a technically and economically competitive fuel when compared to other conventional fossil fuels, such as diesel2）. Biodiesel production from microalgal oil is more promising than other types of oil feedstock microalgae have a fast rate of growth, can be grown in almost all aquatic ecosystems and provide a wide range of different fatty acids which can be used for food, feed and biodiesel applications1, 2）. The other advantages of biodiesel production by microalgae are: i. they can be easily cultivated in seawater, ii. microalgae do not occupied arable lands, so they do not compete with agricultural plants which used as food, iii. they produce high yields of oil per unit area in comparison to oil crops1, 2）. Transesterification is a common method used to convert triglycerides （TG）into biodiesel. This process involves exchanging the organic group of an ester（e.g. TG）with the organic group of an acyl acceptor3, 4）. Carboxylic acids, alcohols, or other esters can be used as acyl acceptors. However, when
alcohol is used as the acyl acceptor, an impure by-product, glycerol（also known as glycerin）, is produced during the transesterification process5）. The glycerin fraction of the transesterification reaction, regarded as spent liquor, can be harmful to the environment6）. An effective method to overcome the glycerin surplus is the transesterification of TG with esters such as methyl or ethyl acetate, producing triacylglycerol instead of glycerol. The mixture of triacylglycerol and fatty acid methyl/ethyl esters can be used directly as biodiesel7）. Biodiesel can be successfully produced using different catalytic processes involving chemical or enzyme catalysts 8）. Enzymatic reaction is of much interest as it is an environmentally friendly process, which produces high purity products9）. Various kinds of acyl acceptors consisting of alcohols and esters can be employed in transesterification reactions catalyzed by lipolytic enzymes. Methanol and ethanol have been the most frequently-used alcohols for the industrial production of biodiesel because of their price and availability. But such types of alcohols have been reported to have a stronger denaturing action on lipase than long-chain aliphatic alcohols10）. The degree of inactivation seems to be inversely proportional to the number of
＊
Correspondence to: F. Tabandeh, Department of Industrial and Environmental Biotechnology, National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran, Omid Tavakoli, School of Chemical Engineering, College of Engineering, University of Tehran, 16 Azar Street, Tehran, Iran. E-mail: [email protected], [email protected] Accepted September 1, 2014 (received for review May 26, 2014)
Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
http://www.jstage.jst.go.jp/browse/jos/ http://mc.manusriptcentral.com/jjocs
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R. S. Alavijeh, F. Tabandeh, O. Tavakoli et al.
carbon atoms of the alcohol used in the transesterification reaction11）. The most common strategy to diminish the risk of enzyme deactivation is stepwise addition of alcohol12−14）. The use of different acyl acceptors such as methyl/ethyl acetate is an alternative and effective way to overcome this problem, because a short-chain alcohol is substituted with the short-chain alkyl acetate, which has no adverse effects on enzyme activity15）. Various acyl acceptors have been employed for transesterification of vegetable oils to biodiesel16−18）. Xu et al.16） suggested using methyl acetate for biodiesel production from renewable oils in a solvent-free medium. In fact, the production of ethyl esters is preferred to methyl esters because ethyl esters have lower pour and cloud points and higher flash and combustion points, so they increase heat content and the cetane number of biodiesel18, 19）. The present study focuses on the main process variables affecting the enzymatic transesterification reaction in the presence of ethyl acetate as an acyl acceptor for the production of biodiesel from microalgal oil as a suitable feedstock. The effect of ethyl acetate on the stability of commercial immobilized lipase, Novozym 435, was also investigated. Crude microalgal oil was employed in this study because the oil purification processes impose extra expenses and increase the final cost of the product.
2 Materials and methods 2.1 Materials Candida antarcitica lipase B（Novozym 435）, immobilized on macroporous acrylic resin with a known activity of approximately 10,000 PLU/g （propyl laurate units/g） , was a gift sample from Novo Nordisk（ A.S., Denmark-Tehran Office） . The powdered form of Chlorella vulgaris was obtained from Qeshm Sina Microalgae Company （Iran） . Standard fatty acid esters were used as reference and purchased from Sigma Aldrich（USA）. All other reagents and solvents used were of analytical grade. 2.2 Oil extraction First, 10 g of microalgal powder was mixed with 150 ml of distillated water. The mixture was sonicated at 70 amplitude for 15 min （Sonicator, Hielscher, Germany） in order to disrupt the microalgae cell wall. Then, a bi-phase solvent solution of chloroform-methanol（1:1 v/v）was added and the mixture was vigorously shaked for 5 min. The resulting mixture was centrifuged at 6708×g for 10 min. The upper polar phase was discarded and the lower phase containing the chloroform and microalgal lipid was separated for further use. Chloroform was evaporated in a hood under ambient temperature20）. The yield of extraction from microalgal oil was 12％.
2.3 Transesterification reaction Novozym 435 was added to mixtures of crude microalgal oil（0.25 g）and ethyl acetate, at different molar ratios of 12:1, 13:1, 14:1, 15:1, 20:1, 25:1（ethyl acetate to oil）. The molecular weight of oil was calculated based on its fatty acid content, obtained by gas chromatography. Different amounts of enzyme（30, 40, 50, 60％ w/w of oil）were then added, and the resulting mixtures containing the reactants were placed in a shaker incubator at different temperatures （30, 35, 40, 45℃） , with shaking rate of 200 rpm for 48 h or 72 h. At the end of the reaction, the enzyme was separated by filtration, the unreacted ethyl acetate was distilled into a concentrator（ Eppendorf Concentrator 5301, Germany）and the crude product was analyzed by gas chromatography. Lipase stability was studied by measurement of the fatty acid ethyl ester（FAEE） content following the transesterification reaction. The mean differences in various groups of the obtained data were compared by one-way ANOVA. Pvalues less than 0.05 were considered significant. Statistical calculations were carried out with the Minitab software version 16. 2.4 Analytical methods The fatty acid content of microalgal oil was measured by gas chromatography（GC）21）. The methyl ester present in the reaction mixture was analyzed using a GC-3800CP gas chromatography system（Varian Crop., Netherlands）connected to a cpsil-5CB capillary column（0.32 mm×30 m, Varian, Netherland）. Methyl heptadecanoate was used as an internal standard, 5 mg of which was mixed with 50 mg of crude product and 1 ml of hexane. Subsequently, 1 μl of the resulting solution was injected into the GC. The column temperature was held at 150℃ for 1 min and then subsequently raised to 200℃ at the rate of 20℃ /min, to 207℃ at the rate of 1℃ /min, to 300℃ at a rate of 30℃ /min and finally maintained at this temperature for 24 min. The temperatures for the injector and flame ionization detector （FID）were set at 270 and 300℃, respectively. The fatty acid ethyl ester（FAEE） content, expressed as a mass fraction in percent, is calculated using the following formula: Σ （A−AIS） CIS×VIS ×100 Eq.1 × FAEE％＝ M AIS Where, ΣA is the total peak area from the fatty acid ethyl esters C14:0 to C24:1, AIS is the peak area of the internal standard（methyl heptadecanoate）, CIS is the concentration of the internal standard solution in mg/mL, V IS is the volume of the methyl heptadecanoate solution in mL and M is the mass of the sample in mg. In order to quantify the FAEE content and identify the by-products of the reaction, the crude transesterification product（1.0 g）was dissolved in ethyl acetate-hexane（1:10 v/v）and then loaded on a column（22 cm×1.5 cm）packed with silica gel（15 g, 60 mesh size）. Elution of the ethyl
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ester fraction was accomplished with an ethyl acetate-hexane mixture. The last collected fraction contained the byproduct formed during transesterification. This fraction was then subjected to thin layer chromatography（TLC）to separate and identify its constituents containing FAEE and triacetin 18）.
3 Results and discussion 3.1 Effect of the ethyl acetate to oil molar ratio on FAEE yield Different molar ratios of ethyl acetate to oil, ranging from 12:1 to 25:1, were used. The fatty acid compositions of obtained esters were C16:0 palmitic acid, C18:0 stearic acid, C18:1 oleic acid, C18:2 linoleic acid and C18:3 linolenic acid. The highest amount of FAEE was obtained at an ethyl acetate to oil molar ratio of 13:1 when an enzyme dosage of 40％ （w/w enzyme/oil） was used at 40℃ for 72 h （Fig. 1）. The reaction medium was insoluble and highly viscous at the 12:1 molar ratio. However, no significant difference was observed when the molar ratio of ethyl acetate to oil was increased to 17:1 （p＜0.05） . The FAEE yield was reduced at higher molar ratios because of the dilution of the reaction medium. In other reports, optimum ratios of ethyl acetate to oil were 12:1 and 11:1, respectively, when various vegetable oils were used as the substrate17, 18）. In the present study, crude microalgal oil contained different kinds of complex compounds that include polar lipids wax esters, sterols and hydrocarbons such as tocopherols, carotenoids and terpenes and phytylated pyrrole derivatives such as chlorophylls22）. Therefore, this can be considered as a -viscous substrate, and it seems that higher amounts of ethyl acetate are necessary to improve the transesterification reaction. 3.2 Effect of enzyme dosage on FAEE yield Maximum FAEE percentage yields of 46.16±4.72 and 47.21±2.23 were obtained at a molar ratio of 13:1 and enzyme doses of 50％ and 40％, respectively（Fig. 2）. An optimum enzyme dosage of 10％ has previously been reported in achieving a 92.7％ FAEE yield from the transesterification of sunflower oil by Novozym 43518）. This is much lower than that used for transesterification of microalgal oil in this study, which may be due to the degree of
Fig. 1 Effect of different ethyl acetate to oil molar ratios on FAEE yield during transesterification of microalgal oil catalyzed by Novozym 435 at a dose of 50%, and incubated at 40℃ with shaking rate of 200 rpm for (■) 48 h and (□) 72 h.
Fig. 2 Effect of enzyme dosage on FAEE yield during transesterification of microalgal oil at 40℃ and 200 rpm for 48 h. ( ) 30% enzyme dosage, ( ) 40% enzyme dosage, ( ) 50% enzyme dosage and ( ) 60% enzyme dosage. purity of this crude oil interfering with the enzyme activity. Moreover, the analysis of the fatty acid content of microalgal oil showed that the amounts of saturated fatty acids （C16:0 and C18:0）were more than those in vegetable oils such as sunflower and Jatropha oils（Table 1）. The main fatty acid components of Chlorella vulgaris were found as palmitic acid（40.92％）, stearic acid（2.41％）, oleic acid （21.20％）, linoleic acid（33％）and linolenic acid（2.28％）. Hence, another reason for requiring a higher enzyme
Table 1 Fatty acid composition of methyl esters prepared from microalgal oil. Type of oil
Fatty acid composition (% w/w)
Ref
16:0
16:1
18:0
18:1
18:2
18:3
20:0
20:1
22:0
24:0
Microalgal oil
40.92
2.41
21.20
33.17
2.28
－
－
－
－
－
This study
Sunflower oil
5.70
－
46.50
44.80
－
－
－
－
－
－
18
Jatropha oil
16.40
1.00
6.20
37.00
39.20
－
0.20
－
－
－
18
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dosage may be due to the hydrolyzing of these saturated fatty acids. 3.3 Effect of temperature on FAEE yield The transesterification reaction was carried out at a temperature range of 30℃ to 50℃. As shown in Fig. 3, the FAEE yield increased when temperature increased from 30℃ to 40℃, and decreased when a temperature of 50℃ was used. A significant difference was observed at 40℃ when compared to other experiments（p＜0.05）. Accordingly, 40℃ was selected as the optimum temperature. The optimum temperatures of 50℃ and 40℃ have also been previously reported to achieve FAEE yields of 92.7％ and 91.0％, respectively from the transesterification of sunflower oil and soybean oil in the presence of ethyl acetate and methyl acetate when using Novozym 43518, 23）. 3.4 Time profile of FAEE production The reactants were exposed to optimum thermal,
enzyme dosage and molar ratio conditions and were then sampled at different time intervals. The FAEE yield was found to increase during a time period of 72 h（Fig. 4）, showing a significant difference at 72 h when compared to the preceding intervals（p＜0.05）. Similar studies using ethyl/methyl acetate as acyl acceptors for the transesterification of vegetable oils have also been carried out, however, optimum reaction times much lower than that of the transesterification of microalgal oil of this study have been reported17, 18）. This again may be due to the use of the enzyme dosage as mentioned above, which suggests that the enzyme in the crude and impure media needs a greater span of time to catalyze the reaction. 3.5 Stability of immobilized lipase in the presence of ethyl acetate The effect of ethyl acetate on the stability of Novozym 435 was determined at repeated transesterification cycles. After each cycle, the enzyme sample was removed and washed with cold acetone. It was then applied to the next cycle using fresh substrate. The transesterification reaction was carried out in 7 cycles sequentially. For comparative purpose, the ethanolysis reaction of microalgal oil by Novozym 435（50％）at 40℃ and 200 rpm involving a threestep addition of ethanol was also carried out in repeated cycles. The results showed that the relative yield of FAEE was approximately 100％ in the presence of the acyl acceptor; ethyl acetate, after 7 cycles, while it decreased dramatically and reached zero after 3 cycles in the presence of ethanol（Fig. 5）. This indicated that ethyl acetate has no
Fig 3 Effect of temperature on ethyl ester yield during interestrification of microalgal oil using ethyl acetate to oil molar ratio of 13:1 at 200 rpm for 48 h.
Fig 4 Time profile of FAEE production during transesterification of microalgal oil catalyzed by Novozym 435 (50% based on oil weight) at 40℃ and 200 rpm, using an ethyl acetate to oil molar ratio of 13:1.
Fig 5 Operational stability of lipase during the transesterification reaction of microalgal oil at 40℃ and 200 rpm using 50% Novozym 435. ( □) Transestrification by ethyl acetate, the molar ratio of ethyl acetate to oil was 13:1 and duration of the transesterification reaction was 72 h. (■) Ethanolysis, for the ethanolysis reaction, the molar ratio of ethanol to oil was 3:1 (three-step addition) during 48 h of the reaction process.
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adverse effects on the enzyme and can be used in largescale for further studies. Modi et al.18） have also studied the stability of Novozym 435 in the presence of ethyl acetate and sunflower oil as substrates. They have shown that lipase is stable in all ten cycles during the transesterification reaction.
4 Conclusion One of the challenges in the commercial production of biodiesel by enzymatic transesterification is the high cost of enzymes, which resulted to elevate the price of the final biodiesel product. Therefore, maintenance of lipase activity during the production cycles is essential. In addition, using a variety of vegetable oils as fuel and energy sources, ultimately leads to interactions with the food chain. Hence, microalgal oil has been suggested as an alternative feedstock for biodiesel production. Microalgae can be grown under different conditions and do not require fertile land and rainy weather. Furthermore, they store high levels of lipid, thus making them appropriate sources of fuel. The results of this study have established ethyl acetate as an acyl acceptor and microalgae as a source of triglycerides for the biodiesel production process. In fact, ethyl acetate has no negative impact on the operational lipase activity during the transesterification reaction, and also produces the valuable by-product, triacetin （glycerol triacetate）, that has widespread applications in the food, printing, tanning, cigarette, cosmetic, pesticide and pharmaceutical industries. Triacetin can also be used as a fuel additive, in the form of an antiknock agent, which can reduce knocking in gasoline engines and improve cold flow and viscosity properties of biodiesel. Also, under the optimized conditions of process variables, and with higher enzyme activities（higher than Novozym 435）, the enzymatic production of biodiesel becomes competitive with the conventional processes that involve acid and alkaline catalytic reactions.
Acknowledgements The work described in this paper was supported by NIGEB（Grant No. 429）and Iran National Science Foundation（INSF Grant No. 89001407）, and would be appreciated.
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