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Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 243–247

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Journal of Molecular Catalysis B: Enzymatic
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Choline-based deep eutectic solvents for enzymatic preparation of biodiesel from soybean oil
Hua Zhao a,∗ , Cheng Zhang b , Tanisha D. Crittle a
a b

Chemistry Program, Savannah State University, Savannah, GA 31404, USA Department of Mechanical Engineering, Georgia Southern University, Statesboro, GA 30460, USA

a r t i c l e

i n f o

a b s t r a c t
In this study, we introduced choline-based deep eutectic solvents (such as choline chloride/glycerol at 1:2 molar ratio) as inexpensive, non-toxic, biodegradable and lipase-compatible solvents for the enzymatic preparation of biodiesel from soybean oil. Through the evaluation of different eutectic solvents and different lipases, as well as the study of reaction parameters (i.e. methanol concentration, Novozym 435 loading and reaction time), we were able to achieve up to 88% triglyceride conversions in 24 h. The enzyme could be reused for at least four times without losing much activity. Our results indicate that new benign eutectic solvents can be used as substitutes of toxic and volatile organic solvents in the enzymatic production of biodiesel from real triglycerides (such as soybean oil). © 2012 Elsevier B.V. All rights reserved.

Article history: Received 24 May 2012 Received in revised form 31 August 2012 Accepted 11 September 2012 Available online 26 September 2012 Keywords: Biodiesel Deep eutectic solvent Enzymatic transesterification Ionic liquid

1. Introduction Biodiesel (i.e. fatty acid monoesters) is becoming an alternative, renewable and biodegradable fuel for diesel engines and heating systems. Biodiesel can be produced from abundant vegetable oils or animal fats [1–3], or other renewable sources of lipids including oleaginous microbial biomass (such as molds, yeasts and algae) [4,5]. Although chemical methods can be effective for biodiesel production, the enzymatic transesterification method offers many advantages over chemical routes such as mild reaction conditions, low energy demand, low waste treatment, the reusability of enzymes, flexibility in choosing different enzymes for different substrates, also allowing a small amount of water in substrates, etc. [6]. Therefore, the enzymatic transesterification of triglycerides to prepare biodiesel is considered a vital and ‘green’ approach when comparing with acid- or base-catalyzed processes. However, the current lipase-catalyzed methods have encountered some challenges, such as the high cost of enzymes, lipase inactivation by acyl acceptors such as methanol, and lipase inactivation by impurities in crude and waste oils, and the use of volatile organic solvents, etc. [6]. Recent advances in ionic liquid (IL) fields have inspired numerous studies in exploiting ILs as reaction media for biocatalysis [7–9], including the enzymatic production of biodiesel [10–15]. Although high yields have been reported in many cases, the high cost of most

∗ Corresponding author. Tel.: +1 912 358 4448. E-mail addresses:, (H. Zhao). 1381-1177/$ – see front matter © 2012 Elsevier B.V. All rights reserved.

ILs prevents these promising ionic solvents from being developed at large scales. Alternatively, a new type of solvents formed by mixing a solid organic salt (such as choline chloride) and a suitable complexing agent (such as urea or glycerol), so called ‘deep eutectic solvents’ (DES), has recently been discovered and employed in a number of applications such as solvents for electrodeposition and electropolishing of metals, catalyst or solvents for chemical and enzymatic reactions, and solvents for extractions [16–19]. Although it is still ambivalent on whether these DES should be formally classified as ILs because they contain a substantial portion of molecular components, they definitely possess many attractive solvent properties as regular ILs. The major advantages of many DES include low-cost, high biodegradability and low toxicity. In relevance to this study, several studies have demonstrated the high enzyme activity and stability in DES, including Candida antarctica lipase B (CALB), C. antarctica lipase A (CALA), and epoxide hydrolase in DES based on choline chloride or ethylammonium chloride and Hbond donors (such as acetamide, ethylene glycol, glycerol, urea, and malonic acid) [20], potato epoxide hydrolase StEH1 in solutions of eutectic mixtures (choline chloride/ethylene glycol, choline chloride/glycerol and choline chloride/urea, all in 1:2 molar ratio) [21], and ␣-chymotrypsin in aqueous mixtures of triethylammonium acetate and urea (ratios ranging from 1 M:2 M to 1 M:5 M) [22]. Recently, our group found that new eutectic ILs (such as choline chloride/glycerol at 1:2 and choline acetate/glycerol at 1:1.5) with low viscosities are also biocompatible with immobilized CALB [23], and cross-linked proteases (subtilisin and ␣-chymotrypsin) immobilized on chitosan [24]. We observed the high reaction rates for

244 H. in the case of choline chloride. To demonstrate such a reaction system being feasible for real lipids. porcine pancreas lipase type II (PPL. we initiate the study of biodiesel preparation from soybean oil via the enzymatic transesterification reaction in DES catalyzed by Novozym 435. CH3 CO2 − ). 13 C NMR (D2 O) ı/ppm = 26. CH2 N. optimum pH 7. The enzyme was removed by filtration. The top layer was sampled and centrifuged. 1 H NMR (300 MHz. lipase PS-D I (immobilized on diatomite. The triglyceride conversion of a particular reaction was estimated by comparing the total peak areas of triglycerides after reaction with the initial triglyceride peak areas before reaction.0 ≥ 20. dried over P2 O5 ) was added into a micro-reaction vessel (5 mL total volume).6 mm. (CH3 )3 N).0 microequivalent of fatty acid from triacetin in 1 h at pH 7. (CH3 )3 N. ∼0. The product weighed 14.3.8 Hz). The percent errors were less than 5%.5 mmol lauric acid and 1.1 product # L3126). urea. 9H. protease activity ≥ 7000 u/g. A known amount of lipase was added to this mixture to initiate the reaction. 3.1 g. Lot # 097K1155). CH2 N. product # L4777.28].8 g of choline chloride in 200 mL of water was passed through the acetate-exchanged column dropwise. ≥10. product # 534781). free lipase B from C. 3H.3 Hz). The column employed was a Phenomenex® Kinetex C18 column (100 mm × 4. Lot # LAKAFC0452302R).0 mmol 1-octanol in 10 mL water-saturated isooctane in 1 h at 20 ◦ C. and the clear supernatant was injected into a LC-20AT Shimadzu HPLC equipped with a SPD-20A UV–visible dual-wavelength detector. aliphatic CH2 ). Zhao et al. ethylene glycol (EG). 2 1 U is the amount of immobilized enzyme which forms 1% octyl laurate (GC. The isocratic eluent consisted of 30/70 (v/v) acetone/acetonitrile. Enzymatic transesterification of soybean oil Soybean oil (100 ␮L.9 (s. ∼9 units/mg. 56.0 mL mixture of DES and methanol. glycerol. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 243–247 Novozym 435-catalyzed transesterification of Miglyol oil 812 with methanol in choline acetate/glycerol (1:1.1 (s. 1. Miglyol oil 812 is a mixture of saturated triglycerides made of relatively short-chain fatty acids (C8 and C10 ). achieving a 97% triglyceride conversion in 3 h under optimum conditions [23].3 (s. Materials The following materials were obtained from Sigma–Aldrich: choline chloride (ChOCl). and lipase immobilized in Sol-Gel-AK from Candida cylindracea (≥10 U/g. and were typically between 0. Vegetable oil (soybean oil): 1 H NMR (300 MHz. and Newlase F from Rhizopus niveus (lipase activity ≥ 30.5 and 1. lipase AK 20 from Pseudomonas fluorescens (lipolytic activity at pH 7. D2 O): ı/ppm = 1. Lot # NC0951704). 30 mL of methanol was added to dissolve the reaction mixture under vigorous stirring. Lot # ILPSAB0152305R). at least.g.0 wt%. the reaction vessel was lifted from the oil bath and chilled briefly in an ice bath to condense the volatiles.. Following the former method. The OH-form resin was then thoroughly washed with distilled water to remove NaOH residues. 3 1 U corresponds to the amount of enzyme which liberates 1 ␮mol oleic acid from triolein per minute at pH 7. The resulting clear liquid was dried over P2 O5 at 45 ◦ C for at least 2 weeks. and dried for further use in the reaction. DES can be easily prepared by either thermally treating the admixed precursors [16. m. and 183. To isolate and purify the product for 1 H NMR analysis. The oil droplets were dispersed in the DES/MeOH mixture after gentle agitation at 50 ◦ C.5 Hz). CALB immobilized on acrylic resin (Novozym 435® . The enzyme was thoroughly washed with water and diethyl ether respectively. Distilled water (50 mL) was then mixed with the residue to dissolve DES and glycerol. The anion-exchange resin was flushed with NaOH solution to ensure the complete loading of hydroxide anions on the column. 2. The triglyceride and product peaks were identified by a UV–vis detector at 210 nm and a refractive index detector (Shimadzu RID10). The injection-loop volume was 20 ␮L. 70. The following Amano lipases were kind gifts from Amano Enzyme USA: lipase PS from Burkholderia cepacia (≥30. The efficiency of anion exchange was examined by silver nitrate test of the eluate exiting the column. antarctica (CALB.4 at 37 ◦ C. the remaining reaction mixture along with THF was filtered to retrieve the immobilized enzyme. CH3 CO2 − ). and methanol was evaporated in vacuo. and thus its enzymatic conversion to biodiesel is easier and faster than real lipids containing long-chain fatty acids.52 (t. detection limit of 1 ppm water) at 20 ◦ C.0 g (76% yield). m. 3. optimum temperature 55 ◦ C. THF (4.5 molar ratio). The Tm of choline acetate was reported as 51 ◦ C [26]. and the HPLC analysis gave the initial concentration (peak areas) of triglycerides present in the reaction mixture. To recycle and reuse Novozym 435.29 (56H. 80 ◦ C).06 (m. optimum pH 6.2.8 mL min−1 .87 (9H.18] or by freeze-drying a solution [27.2 (t.2 product # 62279). Lot # ILPSAC0350403R). CH3 CO2 − ). it is evident that this IL is highly hygroscopic and absorbs moisture quickly when exposed to laboratory air. CH2 OH). To accomplish this. lipase immobilized in SolGel-AK from Pseudomonas cepacia (≥40 U/g. optimum temperature 50 ◦ C. Lot # LPSAC0750102).1. lipase PS-C I (immobilized on ceramic.000 U/g. washing with acetic acid proved highly exothermic). 1. Hydranal® Coulomat AG was used as anolyte for the titration. The reaction mixture was sealed and incubated at 50 ◦ C in an oil bath. CDCl3 [ppm]) CH3 ). Four major triglycerides peaks were integrated between 12 and 30 min. a chromatography column was packed with ∼200 g of Amberlyst® A26 hydroxide (OH) form.000 u/g.6 ␮m). CH2 OH). 58. However. 2H. .5 and 37 ◦ C. A typical reaction contained 1. 30–90 units/mg using triacetin. J = 11. 2. we combined choline acetate (or choline chloride) and a given H-bond donor (e. 1 One unit hydrolyzes 1.3 product # 62278). 2H.000 U/g.91 (s. containing a 1. Amberlyst® A26 hydroxide (OH) form. and the choline acetate eluate was collected and dried in an oven at 100 ◦ C for 24 h. the OH-form resin was exchanged to the acetate form by washing the column with concentrated ammonium acetate solutions (Caution: although effective for anion exchange. 15. optimum temperature 45 ◦ C. J = 14.0% (v/v) water. optimum pH 8.000 U/g. After drying the organic layer with sodium sulfate and filtering off sodium sulfate. Next. Experimental 2. particle size 2.000 u/g. IL and DES preparations A column anion-exchange approach [25] was followed to prepare choline acetate (ChOAc) with a high purity. The water contents for all eutectic ILs were determined in triplicate by the Karl Fischer (KF) titration (Mettler Toledo C20X compact Coulometric. The product was then dissolved in CDCl3 for NMR analysis (JEOL ECX-300 MHz). and the flow rate was 0. The ethyl acetate layer was further washed with 50 mL of water to remove traces of DES and glycerol. the product was collected by evaporation of ethyl acetate under vacuum. area percent) from 0. 2. however. Amano lipase A from Aspergillus niger (≥12. glycerol) at an appropriate molar ratio at 60 ◦ C under rigorous agitation for 30 min (or. J = 4. After rinsing out the residual ammonium acetate with distilled water. and 50 mL of ethyl acetate was added to extract the fatty acid monoesters. After a certain reaction time. and 4. product # 62288). A control experiment was done without the addition of any lipase.60 ı = 0.6 (t.20 (s.0.0 mL) was added into the reaction mixture to fully dissolve the triglycerides and fatty acid methyl esters.0.5. All experiments were run in duplicate.

HC = CHCH2 CH2 ). 2. As shown in Fig. Novozym 435 was generally more active in ChOCl/glycerol (1/2) than in ChOAc/glycerol (1/1. m. m. 40 mg Novozym 435. 300 ␮L methanol.02 (10H.29 (64H. enzyme loading and reaction time Fig.5) (1:2) ChOCl/glycerol (1/2) Fig. 2. RCO2 CH2 CH(O2 CR)CH2 O2 CR and vinyl).28 (2H. 3. other lipases gave much lower triglyceride conversions (typically under 35%). 300 ␮L methanol.32 (8H. O2 CCH2 CH2 ). We further examined the impact of methanol concentration on the enzymatic transesterification reaction in two eutectic solvents. 40 mg Novozym 435. aliphatic CH2 ).0 mL (choline chloride/glycerol (1:2) + methanol). On the other hand. Methanol concentraƟon (v%) Fig. At first.1 g soybean oil. while free CALB was slightly less active (75% conversion).76 (4H. and (4) a higher methanol concentration helps the desorption of glycerol from immobilized CALB preventing enzyme aggregation and blockage of lipase active sites. m.30 (7H.02 (20H. 50 ◦ C and 24 h. HC = CHCH2 CH = CH ).d. 5. the optimized methanol concentration must balance these factors. 40 mg Novozym 435. [Reaction conditions: 700 ␮L choline chloride/glycerol (1:2). vinyl).34 (10 H. m. 1% (v/v) water. These observations not only demonstrate the robustness of immobilized CALB.1 g soybean oil.d. lipase. 1. This is likely caused by the high hydrogen-bond basicity of acetate anions in concentrated ionic solutions.] 3. Trial 1 2 3 4 5 6 7 8 9 Lipase 40 mg Novozym 435 8 mg free CALB 15 mg Amano lipase PS-D I 20 mg Amano lipase PS-C I 8 mg Amano lipase PS 10 mg lipase PPL 15 mg Amano Newlase F 10 mg Amano lipase A from Aspergillus niger 40 mg lipase from Pseudomonas cepacia immobilized on Sol-Gel-AK 40 mg lipase from Candida cylindracea immobilized on Sol-Gel-AK 245 90 Triglyceride conversion (%) 81 75 33 31 35 30 24 32 27 80 Triglyceride conversion (%) 70 60 50 40 30 20 10 ChOAc/glycerol (1:1. A similar trend was observed in our earlier studies of biodiesel synthesis in acetate-based ILs [13] and eutectic solvents [23].60 (8H. HC = CHCH2 CH = CH ). 1. 50 ◦ C and 24 h.5) and 28% in ChOAc/glycerol (1/2). Zhao et al. subtilisin protease was found most active in ChOCl/glycerol (1/2). 1% (v/v) water. (2) a higher methanol concentration also lowers the anion concentration (Cl− or OAc− ) in eutectic solvents.28 (7H. 0. the overall impact of methanol concentration is a convolution of several offsetting factors: (1) a higher methanol concentrations leads to lipase denaturation. . 2 compares the transesterification reaction in three different eutectic solvents. The highest conversion (81%) was observed in ChOCl/glycerol (1/2). which could be beneficial to lipase stabilization. 1% (v/v) water. 3. we examined the transesterification of soybean oil catalyzed by different lipases in a 7/3 (v/v) mixture of ChOCl/glycerol (1/2) and methanol (see Table 1). Biodiesel produced from soybean oil: 1 H NMR (300 MHz. CDCl3 [ppm]) ı = 0. Therefore. O2 CCH2 ). HC = CHCH2 CH2 ). 5. t. 2. d. Therefore. t.86 (9H. 2. t.H. CH3 O2 CR). m. The highest triglyceride conversion (81%) was observed for Novozym 435 at 24 h. CH3 ). followed by 47% in ChOAc/glycerol (1/1. 0. A high methanol concentration is known to denature lipases. 4. O2 CCH2 CH2 ). However. 300 ␮L methanol.74 (3H. m. Since Novozym 435 contains 20 wt% [29] 90 80 Triglyceride conversion (%) 70 60 50 40 30 20 10 0 ChOAc/glycerol ChOAc/glycerol (1:1. This finding is in line with our earlier study [13] where Novozym 435 maintained the highest activity for catalyzing the transesterification of Miglyol oil in etherfunctionalized ILs carrying acetate anions. The optimum methanol concentration in ChOCl/glycerol (1/2) was found to be 30% (v/v). 50 ◦ C and 24 h]. however. RCO2 CH2 CH(O2 CR)CH2 O2 CR). 1% (v/v) water. s. the enzyme activity in eutectic solvents depends on both the types of enzyme and substrate. Effect of different eutectic solvents on the transesterification of soybean oil [reaction conditions: 700 ␮L eutectic solvent. Effect of methanol concentration on the conversion of soybean oil [reaction conditions: total solvent volume 1. Results and discussion 3. Effect of different eutectic solvents. where the highest triglyceride conversion was observed in ChOAc/glycerol (1/1. ChOCl/glycerol (1/2) and ChOAc/glycerol (1/1. (8H. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 243–247 Table 1 Transesterification of soybean oil catalyzed by different lipases.64 (6H.1. 0.2. The optimum loading of Novozym 435 was found to be 40 mg/mL (Fig.5). 2.1 g soybean oil. m.5) ChOCl/glycerol (1:2) 10 31 0 20 30 40 50 Reaction conditions: 700 ␮L choline chloride/glycerol (1:2). m. 1. 0. but also indicate the broad substrate specificity of CALB in biodiesel synthesis.5) in this particular reaction. (3) a higher methanol concentration reduces the medium viscosity and minimize the substrate transport limitation. 3). 2.1 g soybean oil. 50 ◦ C and 24 h]. Screening of different lipases and effect of methanol concentration The preparation and physical properties of deep eutectic solvents based on cholinium salts and glycerol were described in details in our previous study [23]. O2 CCH2 ).5) [23]. 1. 2. t. This order is different from the enzymatic transesterification of Miglyol oil.

Prasad. V. Biotechnol. Goto. 10 (2006) 248. 50 ◦ C and 24 h]. Moniruzzaman. Meher. Effect of enzyme loading on the transesterification of soybean oil [reaction conditions: 700 ␮L choline chloride/glycerol (1:2). [3] J. As shown in Fig. However.1 g soybean oil. J. A further extension of reaction time actually decreased the triglyceride conversion. 0. Chisti. [2] L.1 g soybean oil. a higher triglyceride conversion of 88% (vs 81%) was obtained (Run 1 in Fig. M. Biotechnol. J. Energy Rev. Nakashima. Lett. Technol. which is similar to the case of enzymatic transesterification of Miglyol oil [23]. Fukuda. Sheldon.J.1 g soybean oil. Errazu. Adv. Dupont. the triglyceride conversions actually were lowers (Fig. 82 (2007) 775. 90 Triglyceride conversion (%) 80 70 60 50 40 30 20 10 0 0 10 20 30 ReacƟon Ɵme (h) 40 50 Fig. lipase. Sustain. Therefore. 4. or even 10 wt% [30] CALB. S. G. the actual protein concentration at 40 mg/mL loading is about 4–8 mg/mL. The highest triglyceride conversion of 88% was achieved under the optimized conditions. Choline-based eutectic solvents have the advantages of being inexpensive. Marchetti. 300 ␮L methanol. Bioeng. 0. K. Zhao. We observed more aggregations of enzyme beads at these high enzyme loadings. Plechkova. Chem.2% (v/v) water.246 H.R. the product (i. [9] H. excess glycerol and methanol (Novozym 435 floats in between two layers). biodiesel) settles as an upper layer after the reaction while the bottom layer contains DES. Fig. possibly due to the adsorption of glycerol onto porous acrylic beads. Earle. the same immobilized lipase was used four times in the transesterification reaction. Conclusions We have demonstrated that choline-based eutectic solvents can be used as media for the enzymatic preparation of biodiesel from real triglyceride (soybean oil). for the accuracy of HPLC determination. Chem. N. Kamiya. Chem. Naik. Eng. [7] F.A. / Journal of Molecular Catalysis B: Enzymatic 85–86 (2013) 243–247 90 80 100 88 80 79 79 Triglyceride conversion (%) Soybean oil conversion (%) 20 30 40 50 60 70 60 50 40 30 20 10 0 80 60 40 20 0 1 2 3 4 Novozym 435 loading (mg/mL) Fig. D. 3). Rev. H.2% (v/v). Biotechnol. van Rantwijk. 4.S.W. 300 ␮L methanol. [11] M. 107 (2007) 2757. N. Noda. Chem. We further evaluated the reusability of recycled Novozym 435 after the reaction.F.U. It is important to point out that our experiments were performed at milliliter-scale. 81 (2009) 2045.N. Technol. 92 (2001) 405. [6] C. non-toxic and biocompatible of the lipase. 4 illustrates that the transesterification reaction took 24 h to reach completion. [8] M. 3. [5] Y.e. 0. Time course of enzymatic conversion of soybean oil [reaction conditions: 700 ␮L choline chloride/glycerol (1:2). we fully dissolved the reaction mixture in THF upon the completion of reaction. Lee. Biotechnol.C. 50 ◦ C and 24 h. 50 ◦ C and 24 h]. S. Acknowledgement The financial support from a NSF RIA grant is acknowledged. Kanjilal. 5. Food Chem. Renew. Synth. Kondo. A. Run number Fig. resulting in a small decrease in enzyme activity (triglyceride conversion 88% → 79%). Adv. [10] S. which in turn may block the enzyme’s active sites. This suggests that the lipase was highly stable in the eutectic mixture of 70% (v/v) choline chloride/glycerol (1/2) and 30% (v/v) methanol for an extended period of time at 50 ◦ C. Biosci. Biochem. [4] B. A.A.B. 0. 5. K.-C. A likely reason is the enzymatic glycerolysis occurred in the presence of excess glycerol during the extended reaction period. The best conditions identified are 7:3 (v/v) of choline chloride/glycerol (1/2) and methanol. P. 350 (2008) 160. Gamba. Pure Appl. Reusability of Novozym 435 for biodiesel preparation [reaction conditions: 700 ␮L choline chloride/glycerol (1:2). We also observed that when the water content was reduced from 1% (v/v) to 0. 55 (2007) 8995. J. 40 mg Novozym 435/mL solvent. the separation of product can be achieved through a simple decantation. Liu. J. J.V. Agric. . Renew. Chang. Zhao. 0. J. Seddon. 40 mg Novozym 435.-F. R. Sustain. Energy Rev. Lapis.M.V. 1% (v/v) water.M. R. Zhao et al. 1% (v/v) water and 50 ◦ C]. 11 (2007) 1300. 300 ␮L methanol. 5). Sagar. 25 (2007) 294. 85 (2010) 891. At higher enzyme loadings (50–60 mg/mL). 48 (2010) 295.C. Akoh. 29 (2007) 1881. at a larger reaction setup. Catal. and the optimum enzyme loading here does not imply the actual enzyme loading for large-scale applications.2% (v/v) water. Z. Since our experiments were carried out in a small scale (1 mL). S. A. References [1] H. [12] M. Reddy. Shaw. Sunitha. Miguel. 40 mg Novozym 435. J.N.

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