Bioresource Technology 102 (2011) 6336–6339
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A recyclable enzymatic biodiesel production process in ionic liquids Teresa De Diego, Arturo Manjón, Pedro Lozano, José L. Iborra ⇑ Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, P.O. Box 4021, E-30100 Murcia, Spain
a r t i c l e
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Article history: Received 21 December 2010 Received in revised form 16 February 2011 Accepted 16 February 2011 Available online 20 February 2011 Keywords: Ionic liquids Biodiesel Fatty acid methyl ester (FAME) Lipase Transesteriﬁcation
a b s t r a c t Immobilized Candida antarctica lipase B suspended in ionic liquids containing long alkyl-chain cations showed excellent synthetic activity and operational stability for biodiesel production. The interest of this process lies in the possibility of recycling the biocatalyst and the easy separation of the biodiesel from the reaction mixture. The ionic liquids used, 1-hexadecyl-3-methylimidazolium triﬂimide ([C16MIM][NTf2]) and 1-octadecyl-3-methylimidazolium triﬂimide ([C18MIM][NTf2]), produced homogeneous systems at the start of the reaction and, at the end of the same, formed a three-phase system, allowing the selective extraction of the products using straightforward separation techniques, and the recycling of both the ionic liquid and the enzyme. These are very important advantages which may be found useful in environmentally friendly production conditions. Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction Biodiesel is regarded as a promising fuel to partly replace conventional fossil fuels. Chemically, biodiesel is a mixture of fatty acid alkyl esters (FAMEs) which are produced from a broad range of oil materials, such as vegetable oils, animal fats and waste oil. FAMEs are obtained by catalytic transesteriﬁcation of triglycerides with an alcohol (usually methanol or ethanol). Chemical transesteriﬁcation has been used for the industrial production of biodiesel although the process has several drawbacks: it is energy intensive; recovery of the glycerol by-product is difﬁcult; the acidic or alkaline catalyst has to be removed from the product; alkaline waste water require treatment; and both free fatty acid and water interfere in the reaction (Du et al., 2008). Recently, the enzymatic synthesis of biodiesel has been widely described (Antczak et al., 2009; De Paola et al., 2009; Dwiarti et al., 2010; Li et al., 2009; Maceiras et al., 2009; Sotoft et al., 2010; Verdugo et al., 2010). This enzymatic process has many advantages over the chemical methods. These include moderate reaction conditions, a lower alcohol to oil ratio, easier product recovery, and high conversion, while the main drawback is enzyme deactivation as a result of the hydrophilic nature of the alcohol substrate which limits the number of recycling operations that can be carried out. Ionic liquids (ILs) have been shown to be good solvents for many biochemical processes and to have an exceptional ability to stabilize enzymes during continuous operation (Lozano et al., 2003, 2004, 2007). In this respect, the use of ILs for the synthesis of biodiesel using chemical or enzy⇑ Corresponding author. Tel.: +34 868887398; fax: +34 868884148. E-mail address: [email protected]
(J.L. Iborra). 0960-8524/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2011.02.071
matic catalysis has recently been described. In all cases, the assayed ILs were based on the short-chain 1,3-dialkylimidazolium cation. Neto et al. (2007) assayed the synthesis of biodiesel from vegetable oils using a stannous complex as chemical catalyst, but reported a full and rapid deactivation after the ﬁrst cycle of use. Similarly, Bronsted acidic ILs (e.g. 1-butylsulfonic-3-methylimidazolium sulphate) have been assayed as chemical catalysts to produce biodiesel through the transesteriﬁcation of cottonseed oil with methanol at temperatures higher than 150 °C (Wu et al., 2007). As regards enzymatic catalysis, the IL based short-chain 1,3-dialkylimidazolium cation (e.g. 1-butyl-3-methylimidazolium hexaﬂuorophosphate [Bmim][PF6] or 1-butyl-3-methylimidazolium tetraﬂuoroborate [Bmim][BF4]) have been seen to be inappropriate reaction media for the synthesis of biodiesel (Ha et al., 2007; Gamba et al., 2008; Sunitha et al., 2007; Yang et al., 2010). In all cases, the low solubility of triacylglycerides in the assayed ILs resulted in two-phase reaction media that provided low enzymatic activity, making reaction times close to 24 h necessary to reach full conversion of triglycerides into FAMEs. In the present study, we propose a new enzymatic process for producing biodiesel from vegetable oil, using immobilized lipase as catalyst in a reaction system based on ionic liquids with long alkyl-chains moieties in the cation structure, which provide a homogeneous/one-phase reaction medium.
2. Methods Immobilized (NovozymÒ 435) Candida antarctica lipase B (EC 220.127.116.11) was from Novo Spain. Ionic liquids at a purity of 99% were from IoLiTec (Germany). Standard fatty acids (oleic, palmitic,
T.D. Diego et al. / Bioresource Technology 102 (2011) 6336–6339
linoleic, and stearic), fatty acid esters (methylpalmitoleate, methyllinoleate, methylstearate), monoglycerides (monolinolein, monoolein, monopalmitin, monostearin), diglycerides (1,3-dilinolein, 1,3diolein, 1,3-dipalmitin and 1,3-distearin) and triglycerides (trilinolein and triolein) were from Sigma–Aldrich Chemical Co. Solvents and other chemicals were purchased from Merck and were of the highest purity available. 2.1. Transesteriﬁcation reactions Ninety microlitres of methanol (2.21 mmol) and 310 lL of triolein (0.32 mmol) were added to screw-capped vials of 1 mL total volume containing 800 lL of pure 1-hexadecyl-3-methylimidazolium triﬂimide ([C16MIM][NTf2]) or 1-octadecyl-3-methylimidazolium triﬂimide ([C18MIM][NTf2]). The mixture was vigorously shaken to obtain a homogeneous system. The reaction was started by adding 28 mg of Novozym 435 (10%, w/w, based on oil weight) and run at 60 °C in a glycerol bath with shaking. At regular time intervals, 30 lL aliquots were withdrawn and suspended in 470 lL of hexane. The biphasic mixture was vigorously shaken for 3 min to extract all the substrates and products into the hexane phase. Then, 400 lL of the hexane extracts were added to 100 lL of 250 mM ethyl decanoate (internal standard) solution in hexane, and 40 lL of the resultant solution was analysed by HPLC. The volume of each reaction medium was increased up to ﬁve times to facilitate identiﬁcation of the phases at the end of the enzymatic reaction and orbital shaking was selected to avoid damaging of the immobilized derivative. 2.2. HPLC analysis Reaction products were determined by a Shimadzu HPLC equipped with a multi-channel pump (mod LC-20AD) and a DAD detector (mod SPD-M20A), using a LiChroCart Lichrospher RP-18 column form Merck (25 cm length, 4.6 lm internal diameter and 5 lm particle size). Three mobile phases were employed: phase A, composed of acetronitrile and water (80:20, v/v), phase B of acetronitrile only, and phase C, composed of isopropanol and hexane (55:45, v/v). The ﬂow rate was 1.2 mL min 1 and the injection volume 40 lL. The protocol employed for the mobile phase involved a linear gradient of 100% (v/v) A, decreasing to 0% v/v A in 5 min, while phase B was increased up to 100%. Phase 100% B was maintained for 2 min, before decreasing to 50% in 16 min, while phase C was increased to 50%. The ﬁnal mixture (50:50, v/v B/C) was held for 10 min. Finally, the system was restored to its initial conditions for 6 min. Elution proﬁles were monitored at 210 nm. One unit of activity was deﬁned as the amount of enzyme that produced 1 lmol FAME per min. 3. Results and discussion The enzymatic methanolysis of lipase was tested for the synthesis of fatty acid methyl esters (FAMEs) in two hydrophobic ionic liquids at 60 °C: 1-hexadecyl-3-methylimidazolium triﬂimide ([C16MIM][NTf2]) and 1-octadecyl-3-methylimidazolium triﬂimide ([C18MIM][NTf2]). In this type of reaction media, immobilized lipase is able to transform triacylglycerides into biodiesel. In our case, a 98% yield was obtained after 6 h of reaction at 60 °C, when the amount of immobilized enzyme used was 10% w/w with respect to the assayed weight of vegetable oil. The ILs used allowed the selective extraction of both glycerol and biodiesel reaction products, through easy separation techniques, such as liquid– liquid extraction, by washing with water and hexane in two consecutive steps, as well as the recovery of ionic liquid and the biocatalyst, which can be then used in subsequent cycles for the
production of biodiesel, after the addition of alcohol and triglyceride substrates. This enzymatic process is illustrated in Scheme 1. The ﬁrst step is an enzymatic methanolysis reaction, during which the mixture was vigorously shaken to obtain a homogeneous system. The reaction was started by adding Novozym 435 (10%, w/w, based on oil weight) and at 60 °C in a glycerol bath with shaking. At regular time intervals, samples were analysed by HPLC. For the two ionic liquids tested in this work, the reaction mixture was homogeneous, but, when the reaction ﬁnished, a triphasic system was created: an upper layer containing the FAMEs, a middle layer containing glycerol and unreacted methanol, and a lower layer containing both the ionic liquid and the enzyme. There is no residue FAME and glycerol in this layer, because they are not miscible with the ionic liquids used. At the end of the enzymatic methanolysis reaction (second step), the extraction of glycerol was carried out in the same reactor by liquid–liquid extraction with water. Water was added in a 2:1 v/v ratio with respect to the full reaction volume, and a biphasic reaction system appeared, which was maintained by gentle shaking (100 rpm for 5 min at 60 °C). Then, the aqueous fraction was separated easily by decantation. This washing process was repeated 3-times until the full extraction of glycerol. The traces of water present in the reaction may generate same fatty acids by hydrolysis of FAME, but in a new reaction process, as the alcohol is present in very large amount compared to water, the equilibrium between hydrolysis and transesteriﬁcation is in favour of transesteriﬁcation. The third step involved the extraction of biodiesel, which was carried out in the same enzyme reactor, using liquid–liquid extraction with hexane. After the shaking period, the enzyme reactor was introduced into an ice bath (fourth step), for 2 min to lower the temperature to under 20 °C, which led to the solidiﬁcation of the ionic liquid but not the hexane phase containing the biodiesel. Then, the organic solvent phase containing the biodiesel can be separated by simply decantation. The washing process was repeated three-times until full extraction of the biodiesel. Finally, in a ﬁfth step, the remaining enzyme–IL mixture remaining after the extraction of biodiesel was treated by vacuum pressure ( 1 bar) for 5 min at 60 °C to fully eliminate of any residual organic solvent. The enzyme–IL mixture was then ready for a new biocatalytic cycle of biodiesel production. Fig. 1 illustrates FAME production after a 24 h reaction in the two ionic liquids chosen during nine biocatalytic cycles. These results demonstrate the catalytic efﬁciency of the proposed system. As can be seen, the enzyme was able to catalyse the methanolysis reaction with a yield close to 100% during 9 days of operation in ([C16MIM][NTf2]). However, the FAME production using for [C18MIM][NTf2] decreased after the seventh cycle, probably due to the low stabilization power of the high hydrophobicity of [C18MIM][NTF2] produced by the C18 alkyl chains in the cation. When the methanolysis reaction in a solvent-free system without ionic liquid was analysed, a synthetic activity of 11.92 U g 1 IME was calculated and a production yield of 45.46% after 24 h was obtained. These results clearly demonstrated the protective capacity of these ILs towards enzyme deactivation, since they preserve the lipase activity against of the denaturing effect of the aliphatic alcohol (i.e. methanol) and/or the by-product, glycerol. From the literature, it is known that Cal B lipase is unstable in a medium containing a high concentration of methanol, which limits biodiesel production and a multi-stepwise feeding process was required to avoid these limitations (Yang et al., 2010). For the two ILs chosen, the synthetic activity of each enzyme–IL system was continuously tested during consecutive operation cycles. Fig. 2 shows the synthetic activity of Novozyme 435 for every cycle in the two ionic liquids. The highest synthetic activity was obtained for the [C16MIM] cation compared with [C18MIM].
T.D. Diego et al. / Bioresource Technology 102 (2011) 6336–6339
(only 1 st cycle )
5 th STEP
1 st STEP
Hexane + FAMEs
FAMEs GlyOH MeOH*
IL Phase SOLID
2 nd STEP 4 th STEP Cooling on ice bath
Cooling on ice
3 rd STEP Liquid-liquid extraction
MeOH Glycerol in H 2O
Scheme 1. Enzymatic biodiesel process in ionic liquid.
Fig. 1. FAMEs production after several 24 h reaction cycles in the ionic liquids: (grey) [C16MIM][NTf2] and (white) [C18MIM][NTf2].
These results can be explained by an improvement in the transfer rate of substrates to the enzyme in [C18MIM][NTf2] because the viscosity of these ILs increase with the number and length of the alkyl chain of the cation moiety, which is the main reason for the observed decrease in enzymatic activity. The ILs tested provide the enzyme with an adequate microenvironment that allows high activity and continuous reuse at high concentrations of alcohol (methanol:oil molar ratio of 6:1). Additionally, the conversion of FAMEs obtained with the two hydrophobic ILs tested in this study is higher than that obtained in other studies using different ionic liquids (Ha et al., 2007; Yang et al., 2010).
Fig. 2. Synthetic activity of each enzyme–IL system as a function of the number of continuous operation cycles; (d) [C16MIM][NTf2], and (j) [C18MIM][NTf2].
4. Conclusions This work clearly demonstrates that the reaction system proposed is an appropriate medium for the lipase-catalysed transesteriﬁcation reaction. The excellent level of activity is preserved for further catalytic cycles. Finally, the appropriate selection of an ionic liquid that is not miscible with water or organic solvents permits the formation water–ionic liquid and organic solvent–ionic
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liquid biphasic systems, for the selective extraction of reaction products in two consecutive easy steps with a high yield of biodiesel.
Acknowledgements This work was partially supported by the CICYT (Ref. CTQ200800877), SENECA Foundation (Ref.: 11975/PI/09). We thank Ms. Eulalia Gómez López, laboratory technician in the University of Murcia her help in the experiments.
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