Clean Enzymatic Preparation of Oxygenated Biofuels from Vegetable

Research Article pubs.acs.org/journal/ascecg

Clean Enzymatic Preparation of Oxygenated Biofuels from Vegetable and Waste Cooking Oils by Using Spongelike Ionic Liquids Technology Pedro Lozano,*,† Celia Gomez,† Angel Nicolas,† Ramon Polo,† Susana Nieto,† Juana M. Bernal,†,§ Eduardo García-Verdugo,‡ and Santiago V. Luis‡ †

Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Química, Campus Regional de Excelencia Internacional “Mare Nostrum”, Universidad de Murcia, E-30100 Murcia, Spain ‡ Departamento de Química Inorgánica y Orgánica, Universidad Jaume I, Campus del Riu Sec, Avenida Sos Baynant s/n, E-12071, Castellón, Spain ABSTRACT: The biocatalytic synthesis of oxygenated biofuels (fatty acid solketal esters, FASEs) and biodiesel (fatty acid methyl esters, FAMEs) was carried out by both the direct esterification of fatty acids (i.e., lauric, myristic, palmitic, and oleic acids, respectively) with solketal or methanol, and the transesterification of vegetable oils (i.e. sunflower, olive, cottonseeds, and waste cooking oil) with the same alcohols, in hydrophobic ionic liquids (ILs) based on cations with long alkyl side-chains (e.g., octadecyltrimethylammonium bis(trifluoromethylsulfonyl)imide [C18tma][NTf2]). These hydrophobic ILs are temperature switchable ionic liquid/solid phases that behave as sponge-like system. As liquid phases, they are excellent monophasic reaction media for proposed biotransformations with all the assayed fat substrates, e.g. near to 100% yield of fatty acids solketyl esters (FASEs) and fatty acid methyl esters (FAMEs) in 6 h at 60 °C. By using waste cooking oil mixed with free fatty acids as substrate, green biofuels containing either both FAMEs and FASEs (e.g., aprox. 80% FAMEs and 20% FASEs, etc.) can easily be prepared. Moreover, the reaction mixture can be easily fractionated by iterative centrifugations at controlled temperature into three phases, i.e. solid IL, water, and FAMEs + FASEs mixture leading to a straightforward and clean approach allowing the full recovery of the biocatalyst/IL system for further reuse and the simple product isolation. Furthermore, the enzyme did not shown any loss in activity during reuse in these reaction systems after six operation cycles. KEYWORDS: Ionic liquids, Biocatalysis, Oxygenated biofuels, Biodiesel, Sustainable chemistry, Clean chemical processes

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INTRODUCTION The huge increase in the production of biodiesel fuels (e.g. fatty acid methyl esters) over the past decade has caused an excess of the byproduct glycerol in the market. Biodiesel is usually synthesized by transesterification of triglycerides with short chains alcohols (e.g. methanol, etc.) yielding fatty acid methyl esters (FAMEs) and glycerol as byproducts.1 The full conversion of a triglyceride molecule to biodiesel needs three consecutive transesterification reactions, being involved chemical or enzymatic catalysts, as well as an excess of the alcohol to shift the equilibrium toward the product side. Transesterification is a kinetically controlled process, where the yields depend on the catalytic properties of the enzyme (e.g., enzyme source, type of immobilization or chemical modification of the enzyme, etc.).2 Additionally, as much industrial fat substrates also contain free fatty acids (FFAs) (e.g., waste cooking oils), a direct esterification reaction between the FFAs and the alcohol should occurs to obtain the desired biofuel ester product. As the esterification reaction is a thermodynamically controlled © XXXX American Chemical Society

synthesis, product yields are determined by the thermodynamic constant, while the catalyst only mark the feasibility of the process (e.g., enzyme may become inactivated or inhibited and do not reach thermodynamic yield). For this later case, the elimination of byproduct water molecules during biofuel synthesis is very important in order to shift the thermodynamic equilibrium toward the ester product side (e.g., by using molecular sieves dehydrating agents, and/or vacuum conditions, etc.).3 Although biodiesel is considered as a green fuel, and it is successfully produced on an industrial scale, the industrial process is not green, sustainable, or clean. This industrial process is based on the use of chemical catalysts, mainly homogeneous alkaline catalysts like KOH, where the Special Issue: Building on 25 Years of Green Chemistry and Engineering for a Sustainable Future Received: July 7, 2016 Revised: August 14, 2016

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DOI: 10.1021/acssuschemeng.6b01570 ACS Sustainable Chem. Eng. XXXX, XXX, XXX−XXX

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ACS Sustainable Chemistry & Engineering

Figure 1. (A) Scheme of the immobilized lipase-catalyzed synthesis of fatty acid methyl esters (FAMEs) and fatty acid solketyl esters (FASEs), as green oxygenated biofuels biodiesel, from triacylglycerides or free fatty acids, and methanol or solketal, through transesterification or esterification approaches, respectively. (B) SLILs, [C18tma][[NTf2] and [C18mim][[NTf2], as temperature switchable ionic liquid/solid phase used for the synthesis and purification of FAMEs and FASEs.

upper phase), and neither product deodorization nor neutralization steps are required. Thus, the sustainability of the process is clearly increased by minimizing both energy consumption and waste generation, as well as by improving the overall processing costs through the possible marketability of the resulting high-quality glycerol. The application of enzyme immobilization techniques onto solid supports leads to obtain active and stable heterogeneous biocatalysts, suitable for biodiesel synthesis that allows their easy separation and recycling.2,14−16 Enzyme immobilization may produce alterations in their observed activity, specificity or selectivity as results of the enzyme-support interactions, which in most cases leads to the improvement of its catalytic behavior.17,18 Besides, in the case of biodiesel synthesis, the biphasic reaction system resulted from the nonmiscibility of the triglycerides and the methanol substrates is a key feature that limits the efficiency of any catalytic process. Furthermore, for the case of biocatalysis, these reaction systems also cause full and fast enzyme deactivation as a result of the direct interaction between the catalytic protein and the methanol phase.19 On the other hand, as the final reaction products, i.e. glycerol and biodiesel, are not mutually miscible, this phenomenon also leads to the low catalytic efficiency and deactivation of immobilized due to its poisoning as results of the adsorption of the continuously formed glycerol byproduct onto the support surface. In this sense, the use of ultrasound has been described as an useful technique to avoid the adsorption of undesired byproducts onto the enzyme particles.20 However, it was also demonstrated how the catalytic activity of immobilized enzymes after a biodiesel synthetic cycle can be fully recovered by washing enzyme particles with a small amount of t-butanol, which eliminates the adsorbed glycerol molecules in the enzyme microenvironment without any loss in the enzyme activity.21 In this context, it has been demonstrated how highly hydrophobic ILs based on cations with a long alkyl-side chain (e.g., 1-methyl-3-octadecylimidazolium bis(trifluoromethyl-

undesired side reactions with soap formation (i.e. for the case of oil substrates with high FFAs content), and the difficulties for the recovery and purification of glycerol and the removal of inorganic salts remain important problems because they affect yields and involve the consumption of large amounts of energy and water.4 Furthermore, the sustainability of the biodiesel industry in the near future will be directly linked with both the use of nonedible fatty acid sources as substrates,5 as well as the application of valorization strategies for the byproduct glycerol.6,7 In this context, there are several glycerol derivatives, such as glycerol formal and solketal (1,2-isopropylideneglycerol), obtained by ketalization reaction by using acid catalysts,8 which has been reported as an useful fuel additive for enhancing octane number of gasoline.9 New biodiesels formulations based on mixtures of both FAMEs and fatty acid glycerol esters (FAGEs) has been described as very efficient for diesel engines. Biodiesel blends up to 20% volume fraction of FAGE, exhibiting an excellent suitability as liquid fuel (e.g. viscosity, cetane number, adiabatic flame temperature, etc.),10 as it was demonstrated by testing in an automotive engine.11 Pushing toward the development of sustainable synthetic approaches for producing biofuels, both biocatalysts and ionic liquids have been shown as useful tools.12,13 Because of their high catalytic activity and selectivity, the use of enzymes as catalysts for biodiesel synthesis may be regarded as the perfect solution to the problems associated with conventional chemical-catalyzed processes.14 Biodiesel synthesis by transesterification and/or esterification with immobilized lipase catalysis is applicable to both refined and raw plant oils, free fatty acids (FFAs), waste fats from frying, tallow, and other waste fats. A small excess of alcohols (e.g., methanol, ethanol or propanol) provides a high biodiesel yields under mild conditions (20−60 °C). Both triacylglycerides and FFAs can simultaneously be converted to biodiesel since lipase efficiently catalyzes both transesterification and esterification reactions.2,15 Finally, upon completion of the transesterification process, the glycerol (lower phase) is easily separated from the biodiesel (an B


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sunflower oil, cottonseeds oil, or waste cooking oil, were added in different test tubes with a vacuum septum (5 mL total capacity), containing [C18tma][NTf2] (0.443 g), 80 mg MS13×, and 3 mmol of methanol or solketal, respectively. In all cases, a final 1:2 acyl:alcohol molar ratio was assayed in the reaction media. For each reaction, the oil/alcohol/SLIL mixture was previously incubated in an orbital shaker (IKA KS4000i, Germany) for 30 min at 1000 rpm and at 60 °C to melt the SLIL, becoming a fully clear monophasic liquid medium. The reaction was started by adding Novozym 435 (18% w/w with respect the FFA amount), and the reaction system was sealed under vacuum and incubated at 60 °C for 6 h. At regular intervals, 25 μL aliquots were taken and suspended in 475 μL dodecane/isopropanol (95:5, v/ v) solution, and the resulting biphasic mixtures were strongly shaken for 3 min, and then centrifuged at 15 000 rpm for 10 min to extract biodiesel. Finally, 350 μL of dodecane/isopropanol extracts (upper phase) were added to 150 μL of 100 mM tributyrin (internal standard) solution in dodecane/isopropanol (95:5, v/v), and the final solution was analyzed by CG. Biocatalytic Production of Oxygenated Biodiesel Formulations in [C18tma][NTf2]. Reaction media based on mixtures of free fatty acid with waste cooking oil, or cottonseeds oil, were used as model systems for the biocatalytic synthesis of different oxygenated biodiesel formulations containing both FAMEs and FASEs. Into a 5 mL test tube with vacuum septum, waste cooking or cottonseeds oil (885 mg, equivalent to 1 mmol triolein) were mixed with lauric, myristic, or palmitic acid (up to 0.1 mmol for each FFA), and then, the appropriate amount of methanol and/or solketal was added to finally reach a 1:2 acyl:alcohol molar ratio. Reaction media were completed by the addition of [C18tma][NTf2] (45% w/w) and 80 mg MS13×, respectively, and then were incubated in an orbital shaker (IKA KS4000i, Germany) for 30 min at 200 rpm and at 60 °C to obtain a fully clear monophasic system after melting. The reaction was started by adding Novozym 435 (18% w/w with respect the acyl donor), and the reaction system was sealed under vacuum and incubated at 60 °C for 8 h. At regular intervals, 30 μL aliquots were taken and suspended in 470 μL dodecane/isopropanol (95:5, v/v) solution for GC analysis. At the end of the reaction time, the liquid fraction of the reaction medium was fully collected in separate vials for FAMEs and/or FASEs separation, and SLIL recovery. GC Analysis. GC analysis was performed with a Shimadzu GC2010 (Shimadzu Europe, Germany) equipped with a flame ionization detector (FID) and automatic injector. Samples were analyzed on a TRB-BIODIESEL capillary column (10 m × 0.28 mm × 0.1 μm, Teknokroma, Spain), using tributyrin as internal standard, under the following conditions: carrier gas (He) at 30.0 kPa (15 mL/min total flow); temperature program 100 °C, 10 °C/min, 200 °C, 15 °C/min, 370 °C, variable split ratio, (80:1 to 10:1); detector, 370 °C.24 Peak retention times (min) were as follows: tributirin, 6.7; lauric acid, 4.6; myristic acid, 6.4; palmitic acid, 8.2; oleic acid, 9.7; methyl laurate, 3.6; methyl myristate, 5.5; methyl palmitate, 7.4; methyl oleate, 9.0; solketyl laurate, 8.8; solketyl myristate, 10.4; solketyl palmitate, 11.8; solketyl oleate, 12.8. Operational Stability of the Immobilized Enzyme. Enzyme recycling studies were carried out by using a mixture of free fatty acids (i.e. lauric, myristic and palmitic acids, respectively) with cottonseeds oil, as representative example of the assayed model systems for the biocatalytic synthesis of FAMEs and FASEs (see Table 2, entry 14). For the first operation cycle, cottonseeds oil (350 mg, equivalent to 0.395 mmol triolein) were mixed with lauric acid (50 mg, 0.25 mmol), myristic acid (50 mg, 0.21 mmol), and palmitic acid (50 mg, 0.18 mmol) into a 5 mL test tube with vacuum septum. Then, methanol (82 mg, 2.55 mmol) and solketal (145 mg, 1.1 mmol) were added, reaching a final 1:2 acyl:alcohol molar ratio. Reaction medium was completed by the addition of [C18tma][NTf2] (45% w/w), as described above. The reaction was started by adding Novozym 435 (18% w/w with respect the acyl donor), and the reaction system was sealed under vacuum and incubated at 60 °C for 24 h at 60 °C without the presence of MS13×. At this time, a 30 μL aliquot was taken and suspended in 470 μL dodecane/isopropanol (95:5, v/v) solution for GC analysis as described above. After the catalytic cycle, the liquid

sulfonyl)imide, [C 18 mim][NTf 2 ]; octadecyltrimethylammonium bis(trifluoromethylsulfonyl)imide [C18tma][NTf2], etc.) are able to dissolve both triolein and methanol at any concentration, providing one-phase reaction media. The excellent suitability of these media for enzyme-catalyzed biodiesel synthesis by methanolysis of pure vegetable oils, e.g. standard triolein, was demonstrated (e.g. up to 96% methyl oleate yield after 6 h in [C18mim][NTf2] at 60 °C),22,23 including the total preservation of the catalytic activity for subsequent reuse (e.g., up to 1370 days half-life time for Novozym 435 in [C18tma][NTf2] at 60 °C).24 Additionally, an unique feature of these hydrophobic ILs with long alkyl sidechains is the possibility of switching from the liquid phase to a solid phase by cooling at room temperature (e.g., 53 °C mp for [C18mim][NTf2]), showing an spongelike behavior.25 As solid phases, the reaction mixture can easily be fractionated by iterative centrifugations at controlled temperature into three phases, i.e. solid IL, glycerol, and pure biodiesel.24 These spongelike ionic liquids (SLILs) are able to “soak up” methyl oleate as liquid phase and, then, could be “wrung out” by centrifugation in the solid phase, permitting us to develop a straightforward and sustainable approach for producing nearly pure biodiesel as well as the full recovery and reuse of the biocatalyst/IL system for successive cycles.26 The aim of this paper is to demonstrate the suitability of two different SLILs (i.e. [C18tma][NTf2] and [C18mim][NTf2]), as reaction media for Novozym 435-catalyzed FAMEs and fatty acid solketal esters (FASEs) synthesis through both esterification and transesterification reactions with independence on the fatty acid source, and by using methanol or solketal as nucleophiles (see Figure 1). In order to shift both thermodynamic and kinetic equilibria toward biofuel ester products, all assayed reaction media contained an excess of nucleophile acyl acceptor (i.e., methanol and/or solketal), as well as a dehydrating agent, and were carried out under vacuum conditions. In this way, the enzymatic esterification approach was tested by using different free fatty acids (FFAs) (i.e. lauric, myristic, palmitic, and oleic acids) as acyl donor, while the transesterification path was studied by using food-market vegetable oils (i.e., sunflower and olive oil), cottonseed oil, and waste cooking oil. Additionally, biodiesel formulations having different FAMEs/FASEs content were prepared from cottonseed oil and waste cooking oil mixed with FFAs (model systems), being then separated by a straightforward centrifugation protocol based on the spongelike behavior of these ILs.

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EXPERIMENTAL SECTION

Immobilized Candida antarctica lipase B (Novozym 435, EC 3.1.1.3) was from Novozymes S.A. (Spain). Triolein (65% purity, technical grade), cottonsead oil, solketal (1,2-isopropylideneglycerol, 99% purity), molecular sieves 13× (MS13×; 270 mg H2O/g adsorption capacity), solvents, and other chemicals were from Sigma-AldrichFluka (Madrid, Spain). Food-market oils (i.e., olive and sunflower) and waste cooking oil were from local suppliers. The ILs 1-octadecyl3-methylimidazolium bis(trifluoromethylsulfonyl)imide ([C18mim][NTf2], 99% purity; mp 53 °C)27 and octadecyltrimetylammonium bis(trifluoromethylsulfonyl)imide ([C18tma][NTf2], 99% purity; mp 74 °C)24 were obtained from IoLiTec GmbH (Germany). Novozym 435-Catalyzed FAMEs and FASEs Synthesis in SLILs. For the esterification approach, lauric, myristic, palmitic, or oleic acid (1.5 mmol) was added in different test tubes with vacuum septum (5 mL total capacity), containing 0.5 g of SLIL (i.e., [C18mim][NTf2 or [C18tma][NTf2]), 80 mg MS13×, and 3 mmol of methanol or solketal, respectively. For the transesterification approach, 443 mg of olive oil (amount equivalent to 0.5 mmol triolein), C


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ACS Sustainable Chemistry & Engineering fraction of the reaction medium was fully collected in a separate vial, while the remaining immobilized enzyme particles were washed with tbutanol (0.2 mL) and, then, reused for a new operation cycle. Recovery of FAMEs and FASEs from the SLILs. Reaction mixtures obtained from the biocatalytic synthesis of FAMEs and/or FASEs were placed into 2 mL vials, and then incubated at 60 °C until a fully clear and homogeneous phase was observed. Then, hot water (0.5 mL, 60 °C) was added to each sample, and the resulting multiphase solutions were strongly shaken for 10 min at 60 °C, being finally cooled to room temperature. The FAMEs-FASEs/water/SLIL multiphasic mixtures were consecutively centrifuged three times at 15 000 rpm (50 min) and at room temperature (noncontrolled), 25 and 20 °C, respectively, resulting in three phases, as follows: a top liquid phase of FAMEs/FASEs, an aqueous middle phase, and a bottom phase containing the solid IL. The top phases were collected, washed again with 1 mL water at 60 °C for 15 min, and finally centrifuged (30 min at room temperature) to reach full separation between biofuels and any residual SLIL content. The resulting clean FAMEs/FASEs fractions (top phase) were collected and analyzed by GC/MS. The residual IL content in this FAMEs/FASEs phase was determined by NMR, as follows: for each case, an 80 μL sample of top phase was dissolved in 340 μL acetone-δ6 containing TFA (80 μL) as internal standard was prepared. Samples were analyzed by 188 MHz 19F NMR in a Bruker AC 200E spectrometer, and the residual IL was quantified with respect to standard [C18tma][NTf2] solution in acetone-δ6 containing TFA. Identification of FAME and FASE Products by GC/MS. GCMS analyses were carried out by using a GC-6890 (Agilent, USA) coupled with a MS-5973 (Agilent, USA) system. The GC was equipped with an HP-5MS column (30 m × 0.25 mm × 0.25 μm, Agilent, USA). For FAMEs identification, the following conditions were used: carrier gas (He) at 1 mL/min; inlet split ratio, 1:4; temperature program: 125 °C, 8 °C/min, 145 °C, 26 min; 2 °C/min, 220 °C, 2 min. For FASEs identification, the following conditions were used: carrier gas (He) at 1 mL/min; inlet split ratio, 1:1; temperature program: 60 °C, 1 min; 10 °C/min, 290 °C, 5 min; MS source ionization energy, 70 eV; the scan time was 0.5 s, covering a mass range of 40−800 amu. Each FAMEs and FASEs peak was identified by comparison of its mass spectra with those in a computer library (NIST Library). Methyl laurate, retention time (Rt, min): 8.6; positive ion (m/z): 55.1, 74.1, 87.1, 143.1, 171.2, 183.2. Methyl myristate, Rt: 19.1; (m/z): 55.1, 74.1, 87.1, 143.1, 199.2, 242.2. Methyl palmitoleate, Rt: 36.9; (m/z): 55.1, 69.1, 83.1, 97.1, 137.1, 152.1, 180.2, 194.2, 236.2, 268.3. Methyl palmitate, Rt: 39.3; (m/z): 55.1, 74.1, 87.1, 143.1, 227.2, 241.2, 270.3. Methyl linoleate, Rt: 50.1; (m/z): 55.1, 81.1, 109.1, 150.1, 263.3, 294.3. Methyl oleate, Rt: 50.5; (m/z): 55.1, 74.1, 97.1, 123.1, 152.1, 180.2, 222.2, 264.2, 296.3. Methyl stearate, Rt: 51,8; (m/ z): 55.1, 74.1, 87.1, 143.1, 199.1, 255.2, 298.3. Solketyl laurate, Rt: 18.5.0; (m/z): 101.0, 129.1, 299.2. Solketyl myristate, Rt: 20.4; m/z: 101.0, 129.1, 327.3. Solketyl palmitoleate, 22.00; m/z: 101,1, 129.1, 129.1, 355.3. Solketyl palmitate, Rt: 22.1; m/z: 101.1, 129.1, 355.3. Solketyl oleate, Rt: 23.4; m/z: 101.1, 129,1, 185.2, 338.3, 381.0. Solketyl stearate, Rt: 23.6; m/z: 101.1, 129,1, 340.4, 383.4.

Figure 2. Time-course profiles of TAGs (●), DAGs (△), MAGs (▼), FFAs (○), and FAMEs/FASEs(■) for the Novozym 435-catalyzed FAMEs (A) and FASEs (B) synthesis by transesterification of cottonseeds oil with methanol, or solketal, respectively, in 45% w/w [C18tma][NTf2] at 60 °C.

FAMEs (biodiesel) or FASEs (oxygenated biofuels), respectively, in [C18tma][NTf2] at 60 °C. As can be seen, the biocatalytic system was able to produce up to 98% FAMEs (biodiesel) yield in 6 h from this cottonseed oil, a profile similar to the previously reported for the case of triolein.22 The Novozym 435/[C18tma][NTf2] system also showed an excellent performance for transforming the acylglycerides of cottonseeds oil to FASEs. The corresponding FASEs were obtained with up to 98% yield (see Figure 2B) showing a similar time-course profiles than those obtained for methanol case. This fact can be related to the ability of hydrophobic ionic liquid based on cation with long alkyl side chain (e.g. [C18tma][NTf2], [C18mim][NTf2]) to simultaneously dissolve both vegetable oil and alcohols. The resulting monophasic reaction media enhances the reaction efficiency enabling an excellent transport rate of substrates/products in the microenvironment of the immobilized enzyme.22,23 In order to demonstrate this excellent suitability, with independence of the nature of the acylglyceride donor source, Figure 3 shows the resulting FAMEs and FASEs product yields obtained by Novozym 435-catalyzed transesterification reaction between vegetable oils (i.e., sunflower, olive, cottonseed, or waste cooking oils, respectively), and methanol or solketal, as substrates, and by using either [C18tma][NTf2] (Figure 3A) or [C18mim][NTf2] (Figure 3B) SLILs as reaction media. As can be seen, the combination of Novozym 435 biocatalyst with these SLILs leads to highly efficient systems for the production of FAMEs and FASEs. Independently of the nature of either the vegetable oil source or the nucleophile acyl acceptor (methanol or solketal), product yields were for all cases assayed higher than 93%, after 6 h reaction time. The best results (up to 99% FASEs and FAMEs yield) were obtained when using cottonseeds oil substrate in [C18tma][NTf2] reaction media. These excellent results clearly agrees with the previously reported biocatalytic synthesis of methyl oleate by methanolysis of triolein, where both [C18mim][NTf2],22,27 and [C18tma][NTf2],23 were shown as exceptional reaction media for

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RESULTS AND DISCUSION Lipase-Catalyzed FAMEs and FASEs Synthesis from Vegetable Oils in SLILs. The suitability of immobilized Candida antarctica lipase B to carry out the biocatalytic synthesis of FAMEs and FASEs was first studied by a transesterification approach in two different SLILs (i.e., [C18tma][NTf2] and [C18mim][NTf2]) as reaction media at 60 °C. In this way, four different vegetable oils (i.e., sunflower, olive, cottonseeds, triolein, and waste cooking oil) were tested as acyl donor substrates by using either methanol or solketal as nucleophile (acyl acceptor). As representative example, Figure 2 depicts the time course for Novozym 435-catalyzed transesterification of cottonseeds oil with methanol (Figure 2A) and solketal (Figure 2B) as acyl acceptors, to produce D


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composed by FAMEs and FASEs mixtures, and with independence of the fat substrate source, several oils (i.e. cottonseeds and waste cooking) mixed with FFAs (i.e., lauric acid, myristic acid, and palmitic acid up to 30% w/w) were assayed. In the same context, methanol, solketal, or methanol/ solketal mixtures at different molar proportions (1:2 acyl: alcohol final molar ratio) were tested in order to evaluate the simultaneous production of FAMEs and FASEs (see Table 2). As can be seen, excellent yields (higher than 94%) were observed for all the assayed reaction mixtures. By using a mixture of waste cooking oil with FFAs (entries 1−6) as acyl substrates, it was observed an improvement in both FAMEs and FASEs product yield (up to 98.9%). In the same way, the use of cottonseeds oil mixed with FFAs as substrate (entries 7−12) also provides an improvement in product yield by the increase in FFAs content (up to 99.6% for the solketal case). The mixture of methanol and solketal in the reaction media based on waste cooking oil-FFAs or cottonseeds-FFAs as substrates (entries 13−14) also provide excellent results (up to 98.9% overall product yield, entry 14). Thus, an oxygenated biofuel containing ca 80% FAMEs and ca 20% FASEs was directly synthesized when both methanol and solketal were simultaneously present in the reaction media. These results clearly demonstrate the excellent suitability of the proposed Novozym 435/[C18tma][NTf2] systems to synthesize FAMEs and/or FASEs with independence of the nature of vegetable oil source (i.e., composition of triacylglycerides, FFAs content, nature of FFAs, etc.), which results in an excellent tool for the easy production of oxygenated biofuels. However, the recovery and reuse of both the immobilized enzyme and the SLILs are key criteria to be taken into account for any further application, including the sustainability of the process.13,26 In this context, it was previously reported how [C18tma][NTf2] strongly protects the immobilized enzyme toward both the reuse (i.e., up to 15 consecutive cycles without any loss in activity) and the storage under hot conditions (i.e., up to 1370 days half-life time at 60 °C), resulting in an excellent protective reaction media for Novozym 435.24 Concerning the separation of products (FAMEs and/or FASEs), and the recovery of the [C18tma][NTf2] for further reuse, the previously reported protocol, based on the spongelike properties of this ILs,24 has been improved. Taking the reaction mixture of entry 13 (Table 2) as representative example, Figure 4A shows the clear reaction medium that result after the biocatalytic process at 60 °C. This FASEs/FAMEs/ [C18tma][NTf2] monophasic mixture become heterogeneous after the addition of water (Figure 4B), because of its antisolvent effect with respect both the products and the IL. This heterogeneous mixture was strongly shaken for 60 min at 60 °C and, then, was cooled to room temperature The resulting suspension was consecutively centrifuged three times (15 000 rpm, 50 min) at room temperature (noncontrolled), 25, and 20 °C, respectively, resulting in three phases, as follows: a top liquid phase of FAMEs/FASEs mixture, an aqueous middle phase, and a bottom phase containing the solid [C18tma][NTf2] IL, which followed the density parameter (FAMEs/ FASEs < water < SLIL, see Figure 4C). It should be noted how the direct centrifugation (without previous addition of water, Figure 4D) of the semisolid reaction mixture at room temperature also permitted separation between FAMEs/ FASEs products and the solid [C18tma][NTf2], because of the sponge-like character of this IL (see Figure 4E). However, this approach did not provided an appropriate separation,

Figure 3. FAMEs (dark bars) and FASEs (white bars) yield obtained by the Novozym 435-catalyzed transesterification of sunflower oil (S.O.), olive oil (O.O.) cottonseeds oil (C.O.), and waste cooking oil (W.C.O.) with methanol or solketal, respectively, in 45% w/w [C18tma][NTf2] (A) or [C18mim][NTf2] (B) after 6 h reaction time at 60 °C.

enzyme-catalyzed biodiesel synthesis (e.g., up to 100% yield in 8 h at 60 °C), with exceptional enzyme stability.24 Lipase-Catalyzed FAMEs and FASEs Synthesis from Free Fatty Acids (FFAs) in SLILs. One important difference between vegetable oils obtained from different biomass sources concerns the content and nature of FFAs. This fact is key for the classical industrial process of biodiesel production, based on homogeneous alkaline or acid catalysis, because of the generation of undesired soaps.5 In this context, the suitability of the Novozym 435/SLIL systems to synthesize both FAMEs and FASEs by direct esterification of different FFAs (i.e., lauric acid, myristic acid, palmitic acid, and oleic acid) with methanol or solketal, was also studied by using either [C18tma][NTf2] or [C18mim][NTf2] SLILs as reaction media at 60 °C. As can be seen in Table 1, all Novozym 435/SLILs systems were also Table 1. Biocatalytic Synthesis of FAMEs and FASEs by Direct Esterification of Free Fatty Acids with Methanol or Solketal in SLILs at 60°C esterification yield at 6 h (%) SLIL

alcohol

lauric

myristic

palmitic

oleic

[C18tma]NTf2] [C18tma]NTf2] [C18mim]NTf2] [C18mim]NTf2]

methanol solketal methanol solketal

98 98 99 95

99 95 99 95

99 99 99 97

99 99 99 96

shown as exceptional reaction media for this biocatalytic process, reaching product yields higher than 94% for all cases. Although the ability of the immobilized enzyme to synthesize FAMEs or FASEs, as a function with the assayed alcohol (methanol or solketal, respectively), was shown as exceptional for both SLILs, the [C18tma][NTf2] was selected for further experiments because of its mp (74 °C), higher than that of [C18mim][NTf2] (53 °C), which facilitate product separation by centrifugation.24 Production of Oxygenated Biofuels in SLILs. In order to evaluate the suitability of the Novozym 435/[C18tma][NTf2] catalytic system to directly provide oxygenated biofuels, E


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Table 2. Product Yield for the Biocatalytic Synthesis of FAMEs and FASEs Using Vegetable Oils Mixed with FFAs as the Acyl Donor Source (500 mg Overall Amount) and Methanol and/or Solketal As Acyl Acceptors (3 mmol Overall Alcohol Amount) in [C18tma][NTf2] SLILs after 6 h Reaction at 60 °Ca vegetable oils (%, w/w) entry 1 2 3 4 5 6 7 8 9 10 11 12 13 14

C.O.

W.C.O.

L.A.

M.A.

90 80 70 90 80 70

5 5 10 5 5 10 5 5 10 5 5 10 10 10

5 5 10 5 5 10 5 5 10 5 5 10 10 10

90 80 70 90 80 70 70 70

added free fatty acids (%, w/w)

alcohol (%, mol/mol)

P.A.

methanol

10 10

100 100 100

10 10 10 10

solketal

FAMEs

FASEs

94 97 98

ndb nd nd 97 98 99 nd nd nd 97 98 99 22c 20d

100 100 100

10 10 10 10

product yield (%)

100 100 100

70 70

95 96 97 100 100 100 30 30

78c 80d

a

C.O., cottonseeds oil; W.C.O., waste cooking oil; L.A., lauric acid; M.A., myristic acid; P.A., palmitic acid. bNot detected. cWith respect to 98% overall product yield. dWith respect to 99% overall product yield.

Figure 4. Phase behavior of reaction mixture resulted for the biocatalytic synthesis of FAMES and FASEs by using waste cooking oil mixed with lauric, myristic, and palmitic acids (see Table 2, entry 13) in [C18tma][NTf2] after 6 h reaction at 60 °C (A), after water addition (B), and after the subsequent centrifugation protocol (15 000 rpm, 50 min at room temperature, 25 and 20 °C, respectively) (C). Phase behavior of reaction mixture at 20 °C (D) and after the subsequent centrifugation by the same protocol (E). Schematic hypothesis of the structural organization of the solid [C18tma][NTf2] SLIL net, showing the ionic layers interacting with FAMEs/FASEs through the hydrophobic alkyl chains, like to a soaked sponge (F) and the resulting dry sponge nanostructure after wringing out by centrifugation coupled with washing (G).

because product showed residual ILs traces.24,25 Furthermore, the antisolvent effect of water not only favors the separation of FAMEs/FASEs from [C18tma][NTf2] but also provides an easy and sustainable way to separate both glycerol and nonreacted alcohols from the reaction mixture.23 The recovered FAMEs/ FASEs fractions after water addition, cooling and centrifugation were IL-free, as determined by 19F NMR, in agreement with

previously reported results for biocatalytic synthesis of methyl oleate in [C18tma][NTf2].24 The success of our protocol lays on the unique ability of these spongelike ILs to first dissolve hydrophobic compounds (e.g. vegetable oils, FFAs, FAMEs, FASEs, etc.) at temperatures around their melting points (resulting in transparent monophasic liquid phases), allowing then to fractionate the mixture F


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ACS Sustainable Chemistry & Engineering by a straightforward methodology like cooling and centrifugation. This behavior can be explained by its solid structural organization. The structure of these ILs consists of two interpenetrating polar and nonpolar networks, showing a nanoscale heterogeneity.28−31 The nanostructural organization of the liquid/solid temperature switchable SLILs phase (e.g., [C18tma][NTf2], etc.) may be described in term of ordered layers in which the alkyl side chains of the cations provide hydrophobic hole able for “housing” FAMEs/FASEs molecules in solid phase (see Figure 4F). Then, the centrifugation step of the semisolid mixture allows “to wring the soaked sponge”, leading to the separation of FAMEs/FASEs products from the IL (Figure 4G). This unique feature can be exploited to achieve a straightforward and clean method allowing the full recovery of the biocatalyst/IL system for further reuse and the simple FAMEs/FASEs isolation. Although they are in the literature many protocols for synthesis of FAMEs, the sustainable methodologies for the synthesis of FASEs are scarce. Perosa et al.32 have recently reported the synthesis of caprylic, lauric, palmitic, and stearic esters of solketal (FASEs) using p-toluenesulfonic as acid catalyst. Although the methodology is a very efficient process with 80−92% FASEs yields after 4 h reaction at 60 °C, under solvent-free media, it requires an additional step for biofuel purification step in order to eliminate the acid catalyst (up to 5% w/w content). This purification protocol involves an initial neutralization step with a saturated solution of sodium carbonate, followed by a filtration step, and a final extraction with organic solvents (i.e. chloroform:water biphasic system). In comparison, the methodology here reported allows the recovery of the IL and the immobilized enzymatic catalyst, as well as the straightforward isolation of the product, by adding just water and without the need of any of VOC solvent. Enzymatic Cyclic Production of Oxygenated Biofuels in SLILs. In addition to the easy separation of products by a clean approach, as well as the recovery and reuse of SLILs, another key criterion for scaling-up any biocatalytic process for producing oxygenated biofuels is the operational stability of the immobilized biocatalyst. As representative example, the simultaneous biocatalytic synthesis of FAMEs and FASEs, by using cottonseeds oil mixed with FFAs (i.e., lauric acid, myristic acid and palmitic acid up to 30% w/w) and a methanol/solketal mixture as substrates (see Table 2, entry 14), was used as model reaction media for enzyme recycling studies. Figure 5 shows the operational stability profile of the Novozym 435 biocatalyst against operation cycles of reuse for the [C18tma][NTf2] SLIL, as reaction medium. As can be seen, both the FAMEs and FASEs yield were remained unchanged after 6 consecutive cycles of biocatalyst reuse, pointing out the excellent suitability of this reaction medium based for the enzymatic synthesis of oxygenated biofuels. At this point, it should be noted that after each catalytic cycle for biofuel synthesis, the biocatalyst particles were washed with t-butanol, to eliminate any glycerol residue adsorbed onto the support surface, in agreement with previous works.21,24 The excellent ability of ILs to overstabilize enzymes in nonaqueous conditions for continuous reuse has been widely reported,13,22,25,26,33 even under extremely harsh conditions (i.e. scCO2 at 120 bar and at 150 °C).34 In this context, the SLILs systems based on cations with a long alkyl chain were shown as excellent to preserve the catalytic activity of the enzyme under operational conditions during biodiesel synthesis.22,24 Furthermore, it was also reported how this extremely ordered

Figure 5. Overall biofuel (●), FAMEs (dark bars), and FASEs (white bars) yields obtained by Novozym 435 during successive catalytic cycles of 24 h at 60 °C, by using cottonseeds oil mixed with lauric acid, myristic acid, and palmitic acid, and the appropriate amount of methanol and solketal, as substrates, in [C18tma][NTf2] reaction medium. See the Experimental Section for more details.

supramolecular structure of ILs in the liquid phase may also be able to act as a “mold”, stabilizing the active 3D structure of the enzyme in these nonaqueous nanoenvironments, as it was previously reported.13,26,35

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CONCLUSIONS This paper shows for the first time a new straightforward and green strategy to produce oxygenated biofuels, based on FAMEs/FASEs mixtures, by lipase-catalyzed transesterification of several vegetable oils and/or direct esterification of FFAs with methanol and/or solketal in [C18tma][NTf2] sponge-like IL, obtaining product yields higher than 94% for all cases. The excellent suitability of these SLILs for the immobilized enzyme action was also demonstrated by the great operational stability observed. Furthermore, a straightforward and green protocol for clean separation of the biofuel products is developed. By this approach, an IL-free reaction product was easily separated, permitting the straightforward recovery of the SLIL for further reuse. Once again, the spongelike behavior of these hydrophobic ILs based on cations with long alkyl side chains (e.g., [C18tma][NTf2]) is clearly demonstrated. These results open up a new way in sustainable chemistry for green biofuels by using different sources of vegetable oil as substrates. By using spongelike ILs in combination with biocatalysts, as well as a clean separation methodology as described here, the possibility for developing sustainable chemical processes of industrial interest is increased.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Present Address

§ ́ J.M.B.: TAHE Productos Cosméticos, Poligono Industrial Oeste, E-30169, Murcia, Spain.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work has been partially supported by CTQ-2015-67927-R, CTQ-2015-68429-R (MINECO/FEDER), and 19278/PI/14 (Fundación SENECA CARM) grants. G


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Clean Enzymatic Preparation of Oxygenated Biofuels from Vegetable

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