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fermentation Article

Effect of Operating Variables and Kinetics of the Lipase Catalyzed Transesterification of Ethylene Carbonate and Glycerol Ana Gutierrez-Lazaro 1 , Daniel Velasco 1 , Diego E. Boldrini 2 Jesus Esteban 3 ID and Miguel Ladero 2, * ID 1

2 3

*

ID

, Pedro Yustos 1

ID

,

Department of Materials & Chemical Engineering, Chemical Sciences College, Complutense University of Madrid, 28040 Madrid, Spain; [email protected] (A.G.-L.); [email protected] (D.V.); [email protected] (P.Y.) Chemical Engineering Pilot Plant (PLAPIQUI), Scientific and Technological Centre, Universidad Nacional del Sur-CONICET, 8000 Bahia Blanca, Argentina; [email protected] Molecular Catalysis Division, Max Planck Institute for Chemical Energy Conversion, Stiftstrasse 34–36, 45470 Mülheim an der Ruhr, Germany; [email protected] Correspondence: [email protected]

Received: 4 July 2018; Accepted: 2 September 2018; Published: 5 September 2018







Abstract: Glycerol carbonate (GC) is a value-added product originating from the valorization of widely available glycerol (Gly), a side stream from the production of biodiesel. Here we approach the production of this chemical comparing two reactions based on the transesterification of Gly with dimethyl carbonate (DMC) and ethylene carbonate (EC). When using DMC, it was observed that the free enzyme CALB (lipase B from Candida antarctica) gave the best results, whereas Eversa Transform (a genetic modification of Thermomyces lanuginosus lipase) performed better than the rest if EC was the reagent. With the selected catalysts, their immobilized analogous enzymes Novozym 435 and Lypozyme TL IM, respectively, were also tested. Observing that the yields for the reaction with EC were significantly faster, other operating variables were evaluated, resulting the best performance using a closed system, tert-butanol as solvent, a concentration of enzyme Eversa Transform of 3% w/w, a molar excess of EC:Gly of 9:1 and a temperature of 60 ◦ C. Finally, several runs were conducted at different temperatures and molar ratios of EC:Gly, fitting a kinetic model to all experimental data for the reaction catalyzed with Eversa Transform. This model included the bimolecular transesterification reaction of Gly and EC catalyzed by the lipase and a reversible ring-opening polymerization of EC. Keywords: glycerol; glycerol carbonate; Novozym 435; Lipozyme TL 100 L; Eversa Transform 2.0; kinetic model

1. Introduction Valorization of glycerol (Gly) is a blossoming topic of research given the need to make good use of the abundant surplus caused by the development of the biodiesel industry [1], which, in the end, has led to a marked decline in the retail prices in spite of its use in the food, healthcare, pharmaceutical, or tobacco industries [2]. Although the valorization of Gly for energy related purposes has been pursued [3,4], it has not proved as successful as using this compound as a platform chemical owing to its rich reactivity that can lead to a very wide array of products of interest for several industries [5,6]. One of the many products that can be obtained is glycerol carbonate (GC), which has attracted a lot of interest in the last years due to its outstanding physicochemical characteristics. It has been used mainly as a green solvent for purposes that include chemical reaction media, CO2 separation

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with membranes or Li-ion batteries, but also in the formulation of products in the food and cosmetic industry [7]. Several reviews have described extensively the methods of production of GC [7–10], which could be summarized in the following reaction pathways: (i) addition of CO or CO2 to Gly under pressurized conditions; (ii) reaction of urea and Gly under vacuum conditions so as to remove the ammonia generated as by-product; and (iii) transesterification of Gly with organic carbonates (OC). For the latter route, the authors of the present work have conducted extensive research using both dialkyl carbonates like dimethyl carbonate (DMC) [11,12] or cyclic carbonates like ethylene carbonate (EC) [13–16], or more recently, propylene and butylene carbonate [17]. The advantage of this reaction pathway is that alcohols or glycols can be concomitantly produced when dialkyl or cyclic carbonates, respectively, are used as co-substrates along with Gly. Whilst the synthesis of GC from Gly by any of the methods described above has mostly been attained with homogeneous and heterogeneous catalysts, the use of enzymes for this process has received far less attention. Thus far, the reaction between Gly and DMC has been reported in the literature mainly using Novozym 435 (immobilized Candida antarctica Lipase B) [18–21], although some efforts have also been made with lipase from Aspergillus niger supported on magnetic nanoparticles [22]. In addition, enzymes have also been used successfully for the transesterification of Gly with diethyl carbonate [23] or the interesting reaction for the simultaneous production of GC and biodiesel via the reaction of DMC and different oils, thus avoiding the generation of Gly [24–27]. In all cases, the conversions attained were quantitative and with high selectivities to the desired product of almost 100%. The free enzyme Eversa Transform (a genetic modification of Thermomyces lanuginosus lipase) is another product commercialized by Novozymes that has shown very promising applications in transesterification reactions, for it has been used to yield biodiesel from different oils as feedstock [28,29] or to concentrate unsaturated monoacylglycerols from fish oil [30]. Lipozyme TL 100 (Thermomyces lanuginosus lipase) is yet another free enzyme that has been tested for the synthesis of sucrose-6-acetate, an intermediate to the synthesis of sucralose, a sweetener; in addition, its immobilized form Lipozyme TL IM was also tried in this study [31]. The latter has also been reported in interesterification reactions to obtain trans-free fats from canola oil and palm oil oleins or hydrogenated soybean oil blends [32] or in acetylation reactions to obtain eugenyl acetate, with antimicrobial activity [33]. Given the limited miscibility between Gly and organic carbonates [13,15], many authors have tried performing the respective transesterifications in the presence of solvents to favor the mutual solubility of the compounds [34,35]. This approach has also been tried in enzymatic reactions, where the solvation of enzymes is known to affect positively on the activity of enzymes like CALB (lipase B from Candida antarctica) [36]. The aim of this work is to study the synthesis of GC by transesterification of Gly with EC (reaction 1) and DMC (reaction 2) with the liquid lipase preparations CALB, Lypozyme TL 100, and Eversa Transform as well as the immobilized lipase preparations Novozyme 435 and Lypozyme TL IM. A progressive optimization of the operational conditions with the best preparations is performed afterwards, selecting the most productive system to perform its kinetic study, finally. 2. Materials and Methods 2.1. Chemicals The reactants used for experimental work were glycerol (purity > 99.9%) from Fisher Chemical, dimethyl carbonate (purity = 99%) from Acros Organics Ltd. and ethylene carbonate (purity = 99%), supplied by Scharlau. As solvents, tert-butanol (purity = 99%) from Alfa Aesar and tetrahydrofuran (purity > 99.9%) from Fisher Chemical were used. For calibration of the HPLC method, the following were used: methanol (purity > 99.9 %) from Scharlau, ethylene glycol (anhydrous, purity = 99.8%)

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and glycerol carbonate (purity 99%) from Sigma-Aldrich in addition to citric acid (purity = 99%) from Sigma Aldrich. 2.2. Enzymes The following free enzymes were employed throughout the experimental work: CALB (Candida antarctica lipase B) and Lypozyme TL 100 L (Thermomyces lanuginosus lipase) and Eversa Transform 2.0 (lipase from a genetic modification of Thermomyces lanuginosus), which were a kind gift from Novo Nordisk Bioindustry. For stability purposes, the commercial formulation of such free enzymes contains glycerol and water; thus, considering the small amounts of the limiting reactant Gly used in the experiments, HPLC analysis of the enzymes was made so as to calculate accurately the amount of Gly to be added in each of the experiments. It was found that the Gly content was 0.37, 0.23 and 0.03 g/L for Eversa Transform, CALB and Lypozyme TL 100 L, respectively. As for immobilized enzymes, Novozym 435 (Candida antarctica lipase B immobilized on a macroporous acrylic resin) and Lypozyme TL IM (Thermomyces lanuginosus lipase immobilized on silica gel) were tested here. 2.3. Transesterification Runs and Analytical Method Kinetic runs were performed in 50 mL round-bottom flasks magnetically stirred and heated in glycerol baths placed on IKA heating plate RCT basic with controlled magnetic agitation and PDI temperature control, working in initial reaction volumes of 25 mL. After mixing the organic carbonate and Gly, the cosolvent (either THF or tert-butanol) was added till getting only one liquid phase. Afterwards, the addition of the biocatalyst was made after the contents of the flasks reached the desired temperature. Two different operation modes were tried using the reaction flask open to the atmosphere or closed with a lid to check for volume changes before and after the experiments. These volume changes were monitored by volumetric and gravimetric measurements, while chemical composition of the remaining liquid was determined by HPLC as explained in the next paragraph. For runs with free enzyme, samples were withdrawn at reaction volumes of 100 µL at a time and then prepared for analysis in 900 µL of an aqueous solution of 5 g/L of citric acid. For runs using immobilized enzyme, 300 µL were withdrawn and subjected to centrifugation so that 100 µL of the supernatant could be finally sampled and prepared for analysis as described above. Subsequent analysis of samples was performed by a HPLC method following a device and method reported in previous works [14]. Briefly, for the HPLC analysis, a method based on ion-dipole interaction and molecular volume exclusion was employed, with a REZEX H+ 300 × 7.5 mm column for carboxylic acids as stationary phase, placed in an oven at 65 ◦ C, H2 SO4 5 mM in Milli-Q water flowing at 0.5 mL min−1 as eluent, and a refractive index equipment set at 45 ◦ C as detector. 2.4. Statistical Methods Once the most reactive system was chosen, several runs changing two classical operation conditions: temperature and molar ratio of carbonate—in excess—to glycerol. A kinetic model was then proposed on the basis of the temporal evolution of the involved compounds concentrations. This model was fitted to all retrieved data at the same time by using a gradient non-linear regression algorithm (Levenberg-Marquardt) coupled to numerical integration (4th order Runge-Kutta) of the kinetic equations, algorithms that were implemented in software Aspen Custom Modeler v10. The statistical analysis led to optimal values of the kinetic parameters in the equations, together with their error intervals and a series of goodness-of-fit parameters that indicate the adequacy of the kinetic model to represent the change of the system chemical composition with time. The basic goodness-of-fit parameter is SQR: the sum of quadratic residues, which should tend to zero as the fitting improves; Other goodness-of-fit parameters are calculated from it: the residual mean squared error (RMSE) corresponds to the square root of SQR divided by the degrees of freedom, for which, again, a trend to

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zero would be indicative of better fittings; and finally Fisher F, with a tendency to infinity for the best fit, as SQR is in its denominator. They were calculated with these equations:

SQR = N C , − C ,
2 SQR = ∑ Ci,exp − Ci,calc

(1) (1)

i=1

SQR (2) RMSE =r SQR N −K RMSE = (2) N−K ∑N C , 2 ⁄K (3) F = ∑i=1 (Ci,calc ) /K . (3) F= ∑ SQR⁄(N − K) . N ∑i=1 SQR/(N − K) where Ci,exp is the molar concentration of each compound, Ci,calc is the concentration of compound i where Ci,exp is the molar concentration of each compound, Ci,calc is the concentration of compound i estimated by the kinetic model with the optimal values of the kinetic parameters, N is the number of estimated by the kinetic model with the optimal values of the kinetic parameters, N is the number of data, and K is the number of kinetic constants in the model (3, for the proposed model). data, and K is the number of kinetic constants in the model (3, for the proposed model). 3. Results Results and and Discussion Discussion 3. 3.1. Preselection Preselection of of Reaction, Reaction, Enzyme, Enzyme, and and Operating Operating Mode Mode 3.1. 3.1.1. 3.1.1. Selection of of Enzymes Enzymes for for Each Each Reaction Reaction The The three three free free enzymes enzymes CALB, CALB, Lypozyme Lypozyme TL TL 100 100 L, L, and and Eversa Eversa Transform Transform were were tested tested to to investigate investigate their their performance performance at at aa molecular level in the transesterification of Gly with EC and DMC. The experiments considered a molar ratio of OC to Gly 2:1ofand The conditions conditionsselected selectedfor forthis thisscreening screening experiments considered a molar ratio of OC to of Gly 2:1 ◦ C in a closed system, which have been tested previously for the same reactions aand temperature of 60 a temperature of 60 °C in a closed system, which have been tested previously for the same catalyzed potassium methoxide methoxide (Esteban et (Esteban al., 2015a). 1 andFigures 2 show1the conversion of reactions with catalyzed with potassium etFigures al., 2015a). and 2 show the Gly reached of asGly wellreached as the yields to as each the products achieved after 24achieved and 48 hafter for reactions conversion as well theofyields to each of the products 24 and 48Ahand for B, respectively. reactions A and B, respectively.

(a)

(b)

Figure 1. 1. Glycerol Glycerol carbonate carbonate yields yields using using lipases lipases CALB, CALB, Lypozyme Lypozyme TL TL 100 100 LL and and Eversa Eversa Transform Transform Figure in the transesterification of glycerol with (a) EC and (b) DMC. Type of reactor: batch closed system; T in the transesterification of glycerol with (a) EC and (b) DMC. Type of reactor: batch closed system; ◦ °C; C; Carbonate: GlyGly = 2:1; concentration: 3% v/v; solvent: tert-butanol (30% v/v)(30% agitation T= 60 = 60 Carbonate: = enzyme 2:1; enzyme concentration: 3% v/v; solvent: tert-butanol v/v) speed: 400 rpm. 400 rpm. agitation speed:

The results from Figure 1a show that Eversa Transform 2.0, an enzyme formulated by The results from Figure 1a show that Eversa Transform 2.0, an enzyme formulated by Novozymes Novozymes A/S for biodiesel production from waste oil, is the most active free enzyme for the A/S for biodiesel production from waste oil, is the most active free enzyme for the reaction between Gly reaction between Gly and EC, reaching a yield to GC of 42% and 54% after 24 and 48 h, respectively, and EC, reaching a yield to GC of 42% and 54% after 24 and 48 h, respectively, compared to 38% and compared to 38% and 49% for CALB (lipase B from Candida antarctica) after the same periods or 31% 49% for CALB (lipase B from Candida antarctica) after the same periods or 31% and 33% for Lypozyme and 33% for Lypozyme TL 100, an enzyme preparation containing the lipase of Thermomyces lanuginosus, a thermophilic fungus normally found in compost heaps and employed in detergent formulation.

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TL 100, an enzyme Fermentation 2017, 3, x preparation containing the lipase of Thermomyces lanuginosus, a thermophilic fungus 5 of 14 normally found in compost heaps and employed in detergent formulation. Figure 1b depicts that the performance of Eversa Transform Transform in in the case of reaction 2 is definitely much lower, lower, practically practically not not observing observing any any product product in in the the first first 24 24 hh and and reaching reaching only only a conversion conversion of much 4% after after 48 h, which agrees with the fact that no products were observed using the immobilized immobilized form form 4% Lipozyme TL IM when performing this reaction [18]. On the other hand, Lipozyme TL 100 showed a Lipozyme TL IM when performing this reaction [18]. after 4848 h, h, although CALB was by remarkable improvement reaching reaching 15% 15%of ofyield yieldafter after2424hhand and23% 23% after although CALB was far the best for this reaction, obtaining 41% and 54% of yield after the same periods. The latter fact by far the best for this reaction, obtaining 41% and 54% of yield after the same periods. The latter indeed is a isgood indicator of why it is the enzyme fact indeed a good indicator of why it is the enzymeofofchoice choiceofofprevious previousworks works for for this this reaction, although itit was was used used in in its its supported supported form form Novozyme Novozyme 435 435 [18–21]. [18–21]. although For this this reason, reason, with with the the two two best best performing performing enzymes enzymes Eversa Transform and and CALB, CALB, further further time a molar ratio of OC to Gly of 3:1. As experiments were conducted conductedfor forup uptoto100 100hhemploying employingthis this time a molar ratio of OC to Gly of 3:1. Figures 2 and 3 depict, yields to to GC observed were As Figures 2 and 3 depict, yields GC observed were83% 83%and and81% 81%for forreactions reactionsAAand andB, B,respectively. respectively. Bearing these results in in mind, mind, these these long long run run experiments experiments were were extended extended to to commercially commercially available available and CALB, CALB, i.e., i.e., Lipozyme Lipozyme TL TL IM IM and and Novozyme Novozyme immobilized enzymes corresponding to TL 100 L and 435 for for the the two two reactions. reactions. The aforementioned figures display the yields to to product product observed observed with with immobilizedenzymes, enzymes,which whichare are comparatively lower than those the enzyme, free enzyme, is immobilized comparatively lower than those withwith the free whichwhich is more morepresumably than presumably ascribable to the fact thatare there are internal strong internal mass transfer limitations than ascribable to the fact that there strong mass transfer limitations within within the particles of the supported enzyme (it is important to state the amount of active enzyme the particles of the supported enzyme (it is important to state that that the amount of active enzyme in in the solids is very 10 times higher than in liquid enzyme preparations). When observing the solids is very high,high, 10 times higher than in liquid enzyme preparations). When observing results results of reaction these are comparable to those obtained by Jung al. under same conditions of reaction 2, these2,are comparable to those obtained by Jung et al.etunder the the same conditions of of temperature, molar ratio of DMC to Gly of 3:1 and using THF as a solvent after 48 h, where they temperature, molar ratio of DMC to Gly of 3:1 and using THF as a solvent after 48 h, where they reached approximately approximately 49% 49% of of yield yield compared comparedto toour our42% 42%employing employingtert-butanol tert-butanol[18]. [18]. reached

Figure thethe useuse of the Transform and Lipozyme TL IM for Figure 2.2.Comparison Comparisonofof of Eversa the Eversa Transform and Lipozyme TL the IM reaction for the between reaction ◦ C; DMC:Gly = 3:1; glycerol with ethylene carbonate. Reaction conditions: closed system; T = 60 between glycerol with ethylene carbonate. Reaction conditions: closed system; T = 60 °C; DMC:Gly = amount of enzyme: 3% v/v; solvent: tert-butanol (30% v/v)v/v) agitation speed: 400400 rpm. 3:1; amount of enzyme: 3% v/v; solvent: tert-butanol (30% agitation speed: rpm.

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Figure 3.3. 3.Comparison Comparison of the use ofenzymes the enzymes enzymes CALB B(lipase (lipase B from fromantarctica) Candida and antarctica) and Figure of of thethe use use of theof CALB (lipase from Candida Novozyme Comparison the CALB B Candida antarctica) and Novozyme 435 in in the the transesterification transesterification of glycerol glycerol with dimethyl carbonate. Reaction conditions: 435 in the transesterification of glycerol with dimethylwith carbonate. Reaction conditions: closed system; Novozyme 435 of dimethyl carbonate. Reaction conditions: ◦system; °C ; DMC:Gly = 3:1; amount of enzyme: 3% w/w; solvent: tert-butanol (30% v/v) closed T = 60 DMC:Gly = 3:1; amount of enzyme: 3% w/w; solvent: tert-butanol (30% v/v) agitation speed: T = 60 C; v/v) closed system; T = 60 °C; DMC:Gly = 3:1; amount of enzyme: 3% w/w; solvent: tert-butanol (30% agitation speed: 400 rpm. 400 rpm. speed: 400 rpm. agitation

3.1.2. Selection Selection of of Open Open vs. vs. Closed Closed System System 3.1.2.

YYGCGC, ,Volume Volumeloss loss(%) (%)

Throughout the the experiments experiments it it was was observed observed that that the the initial initial 25 25 mL mL reacting reacting mixture mixture underwent underwent Throughout certain degree degree of of volume volume loss; loss; subsequently, subsequently, the the two two most most active active free free enzymes enzymes were were tested tested under under the the certain subsequently, the two most active free were same conditions in and open (without lid) and closed (with lid) systems for comparison. same conditions conditions in in and and open open (without (without lid) lid) and and closed closed (with (with lid) lid) systems systems for for comparison. comparison. Figure 44 represents represents the the yields yields to product and volume losses obtained operating with with Eversa Eversa Figure to product and volume losses obtained yields to product and volume losses obtained operating operating with Eversa Transform in in reaction reaction 111 for for the the open open and and closed closed systems. systems. In In Figure Figure 55 it it can can be be seen seen that that after after 100 100 hh of of Transform Transform in reaction for the open and closed systems. operation, the type of system has been found to exert a slight influence on the performance of Eversa operation, operation, the type of system has been found to exert a slight influence on the performance of Eversa Transformin inreaction reaction1: 1: the the yield yield to to GC GC decreased decreased from from 83% 83% to to 77% 77% and and the the loss loss of of volume volume increased increased Transform Transform in reaction 1: the yield to decreased from 83% to from 4% to 10% when an open system was employed. from 4% to to 10% 10% when when an an open open system system was was employed. employed. 100 100 90 90 80 80 70 70 60 60 50 50 40 40 30 30 20 20 10 10 0 0

YGC YGC Volume loss Volume loss

Open System System Open

Closed System System Closed

Figure 4. 4. Yield Yield to to product product and and volume volume loss loss in in reaction reaction 11 under under an an open open and and aa closed closed system system using using Figure Figure 4. Yield to product and volume loss in reaction 1enzyme under an open and a closed system using °C ; EC:Gly = 3:1; load: 3% w/w; solvent: tert-butanol Eversa Transform. Conditions: T = 60 Eversa Transform. Conditions: T = 60 ◦°C; EC:Gly = 3:1; enzyme load: 3% w/w; solvent: tert-butanol Eversa Transform. T = 60 C; EC:Gly = 3:1; enzyme load: 3% w/w; solvent: tert-butanol (30% v/v) v/v) agitation Conditions: speed: 400 400 rpm. (30% (30% v/v) agitation agitation speed: speed: 400 rpm. rpm.

Figure 55 depicts depicts the the kinetic kinetic evolution evolution of of each each of of the the species species in in the the reaction reaction in in the the (A) (A) open open and and Figure Figure 5 depicts the kinetic evolution of each of the species in the reaction in the (A) open and (B) (B) closed systems, respectively, where it can be seen that the molar amount of carbonate that (B) closed systems, respectively, where it can be seen that the molar amount of carbonate that closed systems, respectively, where it can be seen that the molar amount of carbonate that disappears disappears is is higher higher than than that that due due to to its its reaction reaction with with Gly. Gly. This This fact fact is is more more evident evident in in the the closed closed disappears is higher than that due to its reaction with Gly. This fact is more evident in the closed system, where system, where a fast reduction in carbonate and Gly is observed at the beginning of the process. system, where a fast reduction in carbonate and Gly is observed at the beginning of the process. aHowever, fast reduction in carbonate and Gly is observed at the beginning of the process. However, theaa However, the temporal temporal evolutions of the the products are are faster in the the open open system, suggesting that the evolutions of products faster in system, suggesting that certain shift shift in in the the equilibrium equilibrium towards towards the the products products is is obtained obtained in in this this system system or, or, at at least, least, that that the the certain

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temporal evolutions of the products are faster in the open system, suggesting that a certain shift in the equilibrium towards is obtained in this system or, at least,inthat presence of some Fermentation 2017, 3, xthe products 7 of the 14water presence of some water from the environment could result anthe enhancement of from the environment could result an enhancement of the transcarboxylation activity of the enzyme. transcarboxylation activity of theinenzyme. presence of some water from the environment could result in an enhancement of the transcarboxylation activity of the enzyme. B) Closed system A) Open system 50

50

A) Open system

40 30

50 40

mmoles of component mmoles of component

mmoles of component mmoles of component

50 40

Glycerol Ethylene carbonate Glycerol carbonate Glycerol Ethylene glycol Ethylene carbonate Glycerol carbonate Ethylene glycol

30 20 20 10 10 0

0

40

80

0

40

40 30 30 20 20 10 10 0 0

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time (h)

0

Glycerol Ethylene carbonate Glycerol carbonate Glycerol Ethyleneglycol Ethylene carbonate Glycerol carbonate Ethyleneglycol

B) Closed system

20

40

80

120

60

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100

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time (h)

0 0

20

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60

Figure 5. Evolution of the presence of reactants and products in the reaction mixture of reaction 1 for

time (h) time (h) Figure 5. Evolution of the presence of reactants and products in the reaction mixture of reaction 1 for = 3:1; enzyme load: 3% (A) the open system and (B) the closed system. Conditions: T = 60 °C; EC:Gly ◦ (A) the open and (B)presence the closedreactants system.and Conditions: T = 60 C; EC:Gly = 3:1; enzyme load: Figure 5. system Evolution of the products w/w; solvent: tert-butanol (30% v/v)of agitation speed: 400 rpm.in the reaction mixture of reaction 1 for 3% w/w; solvent: tert-butanol v/v) system. agitation speed: 400 (A) the open system and (B)(30% the closed Conditions: T =rpm. 60 °C; EC:Gly = 3:1; enzyme load: 3%

w/w; solvent: tert-butanol v/v)a agitation speed: 400 rpm. difference of performance as the yield to For reaction 2, Figure 6(30% shows much more significant

90 100 80 90 70 80 60 70 50 60 40 50 30 40 20 30 10 20 0 10

YGC

Volume loss YGC Volume loss

GC

YGC, Volume loss (%) Y , Volume loss (%)

product practically doubled froma41% to 81% when using thedifference closed system with respect to For reaction 2, Figure 6 shows much more significant of performance asthe theopen yield to For reaction 2, Figure 6 amounted shows a much more significant difference of performance as the yield to one,practically while the volume loss 45% with the latter setup in contrast 20%respect with the product doubled from 41% toto81% when using the closed systemtowith toclosed the open product practically doubled from 41% to 81% when using the closed system with respect to the open system. This seems loss to beamounted related to the higher volatility of DMC compared to that EC.with In fact, one, while the volume toto45% with latter setup contrast to of 20% thethe closed one, while thethe volume loss amounted 45% with the the latter inin contrast 20% with closed reduction in concentration of DMC, a reagent, results insetup a lower yield toto GC. This is the verified in system. This seems to be related to the higher volatility of DMC compared to that of EC. In fact, system. This seems be related to concentration the higher volatility of DMC compared to thatrapidly of EC. in In fact, the the trends observed intoFigure 7: the of DMC declines much more an open reduction in the concentration of DMC, a reagent, results in a lower yield to GC. This is verified in reduction system. in the concentration of DMC, a reagent, results in a lower yield to GC. This is verified in trends observed in Figure 7: significant the7:concentration DMC declines much more rapidly in an open system. trends observed in2,Figure the concentration ofcould DMC much more in an open For reaction the volume of loss bedeclines easily ascribable to rapidly the removal of the system. For reaction 2, the significant volume loss could be easily ascribable to the removal of the methanol methanol generated during the reaction, whose boiling point is 64.7 °C with the reaction taking place ◦ C with For reaction the significant volume could be easily ascribable to the taking removal of theat the generated during the2,reaction, whose boilingloss point is 64.7 the reaction at the nearby temperature of 60 °C. Additionally, tert-butanol and DMC have boiling points ofplace 83 and methanol generated during the reaction, whose boiling point is 64.7 °C with the reaction taking place ◦ 90temperature °C, respectively, which could lead to some depletionand of this component, too. points of 83 and 90 ◦ C, nearby of 60 C. Additionally, tert-butanol DMC have boiling at theBased nearby temperature of 60 °C. Additionally, tert-butanol and DMC have boiling points ofthe 83 and on the results described it can be concluded that to obtain best respectively, which could lead to somethroughout depletionSection of this3.1, component, too. 90 °C, respectively, which could lead to some depletion of this component, too. yields to GC without incurring in too much loss of reaction volume, the best approach is to conduct Based on the results described throughout Section 3.1, it can be concluded that to obtain the best Based on the results throughout 3.1, itby canEversa be concluded thatintoaobtain best the transesterification of described Gly with EC (reaction Section 1) catalyzed Transform closedthe reactor, yieldsyields to GC without incurring inintoo much loss of reactionvolume, volume, the best approach isconduct to conduct to GC without incurring too much loss of reaction the best approach is to the system employed in the following sections. the transesterification of Gly with catalyzedbybyEversa Eversa Transform a closed reactor, the transesterification of Gly withEC EC(reaction (reaction 1) 1) catalyzed Transform in ain closed reactor, the system employed in the following sections. the system employed in the following sections. 100

0

Open System

Closed System

Figure 6. Yield to product and volume loss in reaction 2 using an open and a closed system with CALB Open System Closed System as catalyst. Conditions: T = 60 °C; DMC:Gly = 3:1; enzyme load: 3% w/w; solvent: tert-butanol (30% Figure 6. Yieldspeed: to product and volume loss in reaction 2 using an open and a closed system with CALB 400 rpm. Figurev/v) 6. agitation Yield to product and volume loss in reaction 2 using an open and a closed system with CALB °C; DMC:Gly = 3:1; enzyme load: 3% w/w; solvent: tert-butanol (30% as catalyst. Conditions: T = 60 as catalyst. Conditions: T = 60 ◦ C; DMC:Gly = 3:1; enzyme load: 3% w/w; solvent: tert-butanol (30% v/v) agitation speed: 400 rpm.

v/v) agitation speed: 400 rpm.

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mmoles of component

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(A) Open system Glycerol DMC Glycerol carbonate 80Methanol 120

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mmoles of component

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(B) Closed system Glycerol DMC Glycerol carbonate Methanol

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mmoles of component

Glycerol DMC Glycerol carbonate Methanol

30 20

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10 (B) Closed system

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400

mmoles of component

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Glycerol DMC Glycerol carbonate Methanol 80

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time (h)

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Figure 7. Evolution of the presence of reactants and products in the reaction mixture of reaction 2 for

20 20 Figure 7. Evolution of the presence of reactants and products in the reaction mixture of reaction 2 for = 3:1; enzyme load: 3% (A) the open system and (B) the closed system. Conditions: T = 60 °C; EC:Gly (A) the open system and (B) the closed system. Conditions: T = 60 ◦ C; EC:Gly = 3:1; enzyme load: 10 10 rpm. w/w; solvent: tert-butanol (30% v/v) agitation speed: 400 3% w/w; solvent: tert-butanol (30% v/v) agitation speed: 400 rpm. 0

0

3.2. Effect of Variables on the40Transesterification of Glycerol with Ethylene Carbonate 0 80 120 0 40 80

120

3.2. Effect of Variables on the Transesterification of Glycerol with Ethylenetime Carbonate time (h) (h) 3.2.1. Effect of the Solvent Figure 7. Evolution of the presence of reactants and products in the reaction mixture of reaction 2 for

3.2.1. Effect of(A) thetheSolvent open system and (B) the closed system. Conditions: T = 60 °C; EC:Gly = 3:1; enzyme load: 3%

The effect of the solvent is tested for the transesterification of Gly with EC for the most active w/w; solvent: tert-butanol (30% v/v) agitation speed: 400 rpm.

enzyme, i.e.,of Eversa this, the another hydrophilicof solvent The effect the Transform. solvent isFor tested foreffect the of transesterification Gly like withtetrahydrofuran EC for the most has been tested due to the promising performance shown in the past for the transesterification active(THF) enzyme, i.e., Eversa Transform. For this, the effect of another hydrophilic solvent like 3.2. Effect of Variables on the Transesterification of Glycerol with Ethylene Carbonate with DMC [18,19] and compared to tert-butanol, which had already been tested for past the for tetrahydrofuran (THF) has been tested due to the promising performance shown in the 3.2.1. Effect of the Solvent oil with DMC (Seong et al., 2011). The yield to GC after 100 h in terttransesterification of soybean the transesterification with DMC [18,19] and compared to tert-butanol, which had already been tested butanol was 83%of asthe described intested Figurefor3,the whereas only 57% was obtained with THF in the medium Theofeffect is transesterification with EC foryield the most for the transesterification ofsolvent soybean oil with DMC (Seong et of al.,Gly 2011). The to active GC after 100 h underenzyme, the same Curiously, thetheproductivity in the first case (in the first six hours, that is, i.e.,conditions. Eversa Transform. For this, effect of another hydrophilic solvent like tetrahydrofuran in tert-butanol was of 83% asdue described in−1Figure 3, whereas only 57% was obtained with of THF initial(THF) conditions) 6 mmoles of promising GC·h ·L−1performance , while, when using THF the value thisin the has beenistested to the shown in the pastas forsolvent, the transesterification medium under the same conditions. Curiously, the productivity in the first case (in the first six hours, −1 −1 with is DMC [18,19] GC·h and compared to tert-butanol, had already tested for the parameter 5 mmoles ·L , so, possibly, enzyme which deactivation due to been THF happens. − 1 − 1 transesterification of soybean oil with DMC (Seong et al., 2011). The yield to GC after 100 h in tertthat is, initial conditions) is 6 mmoles of GC·h ·L , while, when using THF as solvent, the value of was ofEnzyme 83% as GC described whereas only 57% was obtained with THF medium 1 , so,3,possibly, 3.2.2. butanol Effectisof Concentration this parameter 5the mmoles ·h−1 ·in L−Figure enzyme deactivation due in tothe THF happens. under the same conditions. Curiously, the productivity in the first case (in the first six hours, that is,

−1·L−1,different This was studied by trying three levels of THF loading, namely 3, and 4% w/w. initialeffect conditions) is 6 mmoles of GC·h while, when using as solvent, the2,value of this 3.2.2.Again, Effect the of the Enzymeconducted Concentration runsis were h, after which yields todue GCtoofTHF 60%, 83%, and 85% were parameter 5 mmoles GC·h−1·L−1for , so,100 possibly, enzyme deactivation happens. attained for the concentrations enzyme. As shown in Figure 8, therenamely is a saturating behavior This effect wasthree studied by tryingofthree different levels of loading, 2, 3, and 4% w/w. 3.2.2. Effect of theof Enzyme Concentration of the concentration catalyst, which is observed using a concentration of enzyme higher that attained 3% Again, the runs were conducted for 100 h, after which yields to GC of 60%, 83%, and 85% were w/w. This This effecteffect has was already been in literature for this reactionnamely [18,37]2,or, even in some studied byobserved trying three different levels of loading, 3, and and 4% w/w. for the three concentrations of enzyme. As shown in Figure 8, there is a saturating behavior of the the runs conducted for amounts 100 h, after yields of 60%, 83%, andperformance 85% were cases,Again, it appears thatwere increasing higher ofwhich enzyme cantobeGC detrimental to the of concentration of for catalyst, which is observed using shown a concentration of enzyme higher that 3% w/w. attained concentrations of enzyme. in Figure 8, there is a saturating behavior the reaction [22].the Asthree turbidity is observed when As adding the enzyme, an emulsion should be created This effect already beenofshould observed inplace literature this reaction [18,37] or,and and some cases, ofhas the concentration catalyst, which is observed using a concentration of enzyme higher thatin3% and the enzymatic action take mainlyfor at the liquid-liquid interface itseven surroundings.

60100

40

YGC (%)

YGC (%)

effect has already observedofinenzyme literature for [18,37] or, in some it appearsw/w. thatThis increasing higherbeen amounts canthis bereaction detrimental toand theeven performance of the cases, it appears that increasing100higher amounts of enzyme can be detrimental to the performance of reaction [22]. As turbidity is observed when adding the enzyme, an emulsion should be created and the reaction [22]. As turbidity is observed when adding the enzyme, an emulsion should be created the enzymatic action should take place mainly at the liquid-liquid interface and its surroundings. and the enzymatic action should 80take place mainly at the liquid-liquid interface and its surroundings.

20

80

60

40

0 20

2

3

4

Concentration of Eversa Transform (% w/w) 0

3 4 Figure 8. Effect of the amount of the2 Eversa Transform enzymatic preparation used in the Eversa Transform (% w/w) transesterification of Gly with EC onConcentration the yield toofGC. Conditions: T = 60 °C; EC:Gly = 3:1; solvent: tertFigure Effect amount of the Eversa Transform enzymatic preparation used in the butanol (30%8.v/v); t = of 100the h; agitation speed: 400 rpm. Figure 8. transesterification Effect of theof amount of the Eversa Transform enzymatic preparation used in the Gly with EC on the yield to GC. Conditions: T = 60 °C; EC:Gly = 3:1; solvent: tert-

transesterification of v/v); Glyt with on the yield GC. Conditions: T = 60 ◦ C; EC:Gly = 3:1; solvent: butanol (30% = 100 h;EC agitation speed: 400to rpm. tert-butanol (30% v/v); t = 100 h; agitation speed: 400 rpm.

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3.2.3. Effect of the Molar Ratio of Reactants and Temperature The transesterification of Gly with EC had been tested employing relatively9 low Fermentation 2017, 3, x of 14 molar ratios ranging from 1.5:1 to 3:1, although in the absence of any solvents in all cases [13,14,16,38,39]. The molar 3.2.3. Effect of the Molar Ratio of Reactants and Temperature ratio of reactants may affect the equilibrium conversion and the kinetics of the reaction. Considering The of transesterification of Gly with reactions EC had been employing relatively low catalyzed molar ratiosreactions and the low rates reaction in enzymatic intested contrast with chemically ranging from 1.5:1 to 3:1, although in the absence of any solvents in all cases [13,14,16,38,39]. The also the fact that here this reaction has been performed in the presence of a solvent, thus diluting molar ratio of reactants may affect the equilibrium conversion and the kinetics of the reaction. the concentration oflow therates species, it has decided to in make usewith of larger molar excess of organic Considering the of reaction in been enzymatic reactions contrast chemically catalyzed reactions and also the3:1 facttothat this reaction has performed in the presence of a60, solvent, carbonate ranging from 9:1.here Additionally, thebeen reaction was evaluated at 50, and 70 ◦ C, as this thus diluting the concentration the species, it has been make use of larger molar excess is the temperature range that hasofbeen considered in decided kinetictostudies [11,16]. of organic carbonate ranging from 3:1 to 9:1. Additionally, the reaction was evaluated at 50, 60, and Figure 9 shows that better results are obtained as the molar excess increases from 3:1 to 9:1 at all 70 °C, as this is the temperature range that has been considered in kinetic studies [11,16]. temperatures, which agrees withresults the fact that even larger excesses of 17:1from and3:1 21:1 of at diethyl carbonate Figure 9 shows that better are obtained as the molar excess increases to 9:1 all temperatures, which agrees with the fact that even larger excesses of 17:1 and 21:1 of diethyl to Gly were reported previously as a strategy to reach conversions of 84% or 97% [40,41]. On the carbonate to Gly were reported previously a strategy to reach conversions of 84% or 97% [40,41]. other hand, temperature does not show aas clear positive effect on performance at 100 h reaction time, On the other hand, temperature does not show a clear positive effect on performance at 100 h reaction but there is a sharp increase in initial reaction rate when calculating it for the first 6 h of reaction, time, but there is a sharp increase in initial reaction rate when calculating it for the first 6 h of reaction, as expected. as expected.

78.00 79.00 80.00 81.00 82.00 83.00 84.00 85.00 86.00 87.00 88.00 89.00 90.00 91.00 92.00 93.00 93.00

Figure 9. Effect of the molar ratio of EC to Gly and temperature on the yield to GC. Conditions: enzyme Figure 9. Effect of the molar ratio of EC to Gly and temperature on the yield to GC. Conditions: load: 3% w/w; solvent: tert-butanol (30% v/v); t = 100 h; agitation speed: 400 rpm. enzyme load: 3% w/w; solvent: tert-butanol (30% v/v); t = 100 h; agitation speed: 400 rpm. 3.3. Kinetic Modeling of Enzymatic Transesterification of Glycerol With Ethylene Carbonate Carbonate 3.3. Kinetic Modeling of Enzymatic Transesterification of Glycerol With Ethylene To study the kinetics of this reacting system, the evolution of each of the components has been

Tomonitored study the kinetics of this reacting system, the evolution of each of the components has been from the experiments performed at different values of temperature (50, 60, and 70 °C) and monitored the performed atenzyme different values of(3% temperature (50, 60, and 70 ◦ C) and molarfrom ratio of ECexperiments to Gly (3:1 to 9:1), at a constant concentration w/w). To avoid problems due toof solvent EC(3:1 evaporation seemed to enzyme form a low-boiling point azeotrope), runsTo were molar ratio EC toand Gly to 9:1), (they at a constant concentration (3% w/w). avoid problems in closed reactors provided with seemed an adequate system. due to performed solvent and EC evaporation (they to sampling form a low-boiling point azeotrope), runs were It can be observed that the evolution of EC cannot be explained only on the basis of the performed in closed reactors provided with an adequate sampling system. transesterification reaction. At the same time, both products (GC and EG) are obtained in equimolar It amounts, can be thus observed the evolution of ofEC onlyofon the basis of the avoidingthat a possible further reaction GCcannot with Glybe andexplained a polymerization EC and EG or Gly driven by At the the lipases polymerization). However, ring-opening transesterification reaction. same(condensation time, both products (GC and EG) are obtained in equimolar polymerization of ethylene carbonate drivenreaction by EversaofTransform is possible [42]. amounts, thus avoiding a possible further GC with2.0Gly and a polymerization of EC and EG or Gly driven by the lipases (condensation polymerization). However, ring-opening polymerization of ethylene carbonate driven by Eversa Transform 2.0 is possible [42]. Glycerol + Ethylene carbonate → Glycerol carbonate + Ethyleneglycol

(4)

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r1 = k1 ·nGly ·nEC

(5)

where r1 (mol·L−1 ·min−1 ) is the reaction rate for the Gly transesterification driven by the lipase; k1 (L·mol−1 ·min−1 ) is a second-order kinetic constant, nGly is the molar concentration of Gly and nEC the molar concentration of EC. Ethylene carbonate ↔ [Ethylene carbonate]x

(6)

r2 = k2 ·n2EC

(7)

r3 = k3 ·nPEC

(8)

Here, r2 (mol L−1 ·s−1 ) is the reaction rate for the ring-opening polymerization of EC; and r3 is the inverse reaction (ring-closing depolymerization to EC) (mol L−1 ·s−1 ). Their kinetic constants are k2 (L·mol−1 ·min−1 ) and k3 (min−1 ); nEC is the molar concentration of EC and nPEC is the molar concentration of the polymer, whose production can account for the increasing turbidity with time molar excess of EC and reaction temperature. The suggested model with three reactions happening in parallel was fitted to data from runs performed with different excesses of EC and at temperatures ranging from 50 to 70 ◦ C. Results shown in Figure 10 and Table 1 indicate that the excess of EC only helps slightly the formation of GC and EG. As expected, values for the kinetic constants do not depend on the excess of EC, taking into account the respective absolute errors for such constants. The kinetic model fits perfectly to retrieved experimental data, as indicated by the very high values for Fisher’s F parameter, and very low values of SQR and RMSE, together with the evident goodness of fit shown by lines (calculated with the model) and experimental points in the figure. Finally, the maximum error intervals at 95% confidence for the kinetic constants are very small, rendering very narrow confidence intervals for such constants. Table 1. Kinetic and goodness-of-fit parameters for the proposed model and the different runs. Parameter

Run 1

Run 2

Run 3

Run 4

Run 5

k1 k2 k3 SQR RMSE F-value

0.0170 ± 0.0006 0.0046 ± 0.0007 0.0068 ± 0.0033 0.000336 0.0105 23140

0.0174 ± 0.0006 0.0058 ± 0.0007 0.0132 ± 0.0016 0.0000518 0.012 51179

0.0193 ± 0.0011 0.0032 ± 0.0013 0.0082 ± 0.0008 0.000552 0.0012 113139

0.0284 ± 0.0022 0.0089 ± 0.0013 0.0036 ± 0.0024 0.0018 0.021 6883

0.058 ± 0.0051 0.019 ± 0.008 0.0012 ± 0.0006 0.00838 0.017 4883

Common conditions: enzyme load: 3% w/w; solvent: tert-butanol (30% v/v); closed stirred batch reactor; agitation speed: 400 rpm; T = 50 ◦ C for runs 1, 2 and 3; EC:Gly 3:1 molar ratio for runs 1, 4, and 5.

From the kinetic model, it can be inferred that the effect of increasing temperature would seem to affect both parallel reactions (transesterification and the couple polymerization-depolymerization of ethylene carbonate). Results of the fit are shown in Figure 11 and Table 1, and they indicate that temperatures as high as 70 ◦ C affect all reaction rates dramatically, while 50 and 60 ◦ C lead to very similar results (slightly higher reaction rates for 60 ◦ C). In general, these observations correspond to the relatively high activation energies for transesterification (55.21 kJ·mol−1 ), polymerization by ring-opening (63.88 kJ·mol−1 ) and depolymerization by ring-closing (77.96 kJ·mol−1 ). It is interesting to observe that the latter reaction tends to disappear at high temperatures, so the lipase effectively does not act as a good catalyst at high temperatures for this reaction in particular. It is unusual to observe negative activation energies, a phenomenon that can be related to a notably negative enthalpy change with temperature [43], to protein folding dynamics [44] or to deactivation or denaturation of the enzyme, which can happen at least partially [45]. In this latter case, the effect could affect only some of the reactions catalyzed by the enzyme. It should be considered that all kinetic constants here calculated include the activity of the enzyme towards the reaction under consideration.

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Fermentation Fermentation2017, 2017,3,3,xx

12 12 of of 15 15 3.5 3.5

0,3 0.3 0,3 0.3

EC:Gly EC:Gly3:1 3:1 EC:Gly EC:Gly6:1 6:1 EC:Gly 9:1 EC:Gly 9:1

3.0 3.0

3.0 3,0 3.0 3,0

2.5 2.5 2.5

2,5 2.5 2,5

2.0 2.0 2.0 2,0

0,2 0.2 0,2 0.2

2.0 2,0

1.5 1,5 1.5 1.5 1,5 1.5 1.0 1,0 1.0 1.0 1,0 1.0

0,1 0.1 0,1 0.1

0,5 0.5 0,5 0.5 0,0

0,0 0.0 0,0 0.0 0

20 20

0

C 0.4 C 0.4 0,4 0,4

60 60

80 80

0.0 0,0 0.0 0.0 0.0 00

100 100

20 20

40 40

60 60

80 80

100 100

80 80

100 100

time time(h) (h)

time time(h) (h)

D 0.4 D 0.4 0,4 0,4

EC:Gly EC:Gly3:1 3:1 EC:Gly EC:Gly6:1 6:1 EC:Gly 9:1 EC:Gly 9:1

0.3 0,3 0.3 0,3

EC:Gly EC:Gly3:1 3:1 EC:Gly EC:Gly6:1 6:1 EC:Gly 9:1 EC:Gly 9:1

CCEG (mol/L) (mol/L) EG C EG(mol/L) EG

(mol/L)

0.3 0,3 0.3 0,3

40 40

C CG CG(mol/L)

(mol/L) CC CG(mol/L) CCG (mol/L)

B B

3.5 3,5 3.5 3,5

(mol/L) CEC (mol/L) C (mol/L) EC C (mol/L) CC (mol/L) C (mol/L) EC EC EC EC

EC:Gly EC:Gly3:1 3:1 EC:Gly EC:Gly6:1 6:1 EC:Gly EC:Gly9:1 9:1

gly gly (mol/L) CCglygly(mol/L)

(mol/L) CC (mol/L)

AA 0.4 0,4 0.4 0,4

0,2 0.2 0,2 0.2

C

0,2 0.2 0,2 0.2

0,1 0.1 0,1 0.1

0,1 0.1 0,1 0.1

0,0

0,0 0.0 0.0 00

20 20

40 40

60 60

80 80

100 100

0,0

0,0 0.0 0.0 00

20 20

40 40

60 60

time time(h) (h)

time time(h) (h)

Figure 10. the kinetic model proposed toto runs 1,1,1, 2,2,2, and 3 3(molar ratios EC:Gly 1:3, 1:6, and 1:9, Figure 10. Fitting the kinetic model proposed runs and ratios EC:Gly 1:3, 1:6, and Figure 10.Fitting Fittingofof of the kinetic model proposed to runs and 3(molar (molar ratios EC:Gly 1:3, 1:6, and respectively). Subfigure (A) shows results for glycerol, (B) for ethylene carbonate, (C) for glycerol 1:9, respectively). Subfigure (A) shows results for glycerol, (B) for ethylene carbonate, (C) for glycerol 1:9, respectively). Subfigure (A) shows results for glycerol, (B) for ethylene carbonate, (C) for glycerol carbonate, and (D) for ethylene glycol. Other conditions: carbonate, enzyme load: 3% w/w; solvent:tert-butanol tert-butanol carbonate,and and(D) (D)for forethylene ethyleneglycol. glycol.Other Otherconditions: conditions:enzyme enzymeload: load:3% 3%w/w; w/w;solvent: solvent: tert-butanol ◦ °C . (30% v/v); closed stirred batch reactor; agitation speed: 400 rpm; T = 50 (30% v/v); 400 rpm; rpm; TT ==50 50°CC. . (30% v/v);closed closedstirred stirred batchreactor; reactor; agitation agitation speed: speed: 400 1,6 1.6 1,6 1.6

50 50ºC ºC 60 60ºC ºC 70 70ºC ºC

BB 50 50ºC ºC 60 60ºC ºC 70 70ºC ºC

1,4 1.4 1,4 1.4 1,2 1.2 1,2 1.2

(mol/L) (mol/L) CCEC EC EC (mol/L) CC(mol/L)

0.3 0,3 0.3 0,3

1,0 1.0 1,0 1.0

0.2 0,2 0.2 0,2

EC

0.8 0,8 0.8 0,8 0.6 0,6 0.6 0,6

gly

C gly(mol/L) (mol/L) C (mol/L) CCgly gly(mol/L)

AA 0.4 0,4 0.4 0,4

0,1 0.1 0,1 0.1

0.4 0,4 0.4 0,4 0.2 0,2 0.2 0,2

0,0 0.0 0,0 0.0 00

20 20

40 40

60 60

80 80

0.0 0,0 0.0 0,0

100 100

00

20 20

40 40

time time(h) (h) 0,4 0,4

C C

80 80

100 100

D D 0.4 0,4 0.4 0,4 0,3 0.3 0,3 0.3

(mol/L) CCCG CG(mol/L)

C EG (mol/L) EG (mol/L) CCEG EG(mol/L)

(mol/L)

0,3 0,3

0,2 0.2 0,2 0.2

C

0,2 0,2

0,1 0,1

0,0 0,0 00

60 60

time time(h) (h)

50 50ºC ºC 60 60ºC ºC 70 ºC 70 ºC 20 20

40 40

60 60

time time(h) (h)

80 80

100 100

0,1 0.1 0,1 0.1

0,0 0.0 0,0 0.0 0

0

50 50ºC ºC 60 60ºC ºC 70 70ºC ºC 20 20

40 40

60 60

80 80

100 100

time time(h) (h)

◦ C,5560 ◦ C, ◦ C, Figure 11. Fitting kinetic model proposed to runs 1,1, 54,4,(50 and (50 °C , , 60 , , and Figure 11. Fitting of the the kinetic model proposed to1, runs and (50 °C 60 °C and 70 70 °C °C,, Figure 11. Fitting of of the kinetic model proposed to runs 4, and and 70°C respectively). respectively). Subfigure (A) shows results glycerol, (B) (C) respectively). Subfigure (A) shows results for ethylene glycerol,carbonate, (B) for for ethylene ethylene carbonate, (C) for for glycerol glycerol Subfigure (A) shows results for glycerol, (B) for for (C) forcarbonate, glycerol carbonate, and (D) carbonate, and (D) for ethylene glycol. Other conditions: enzyme load: 3% w/w; solvent: tert-butanol carbonate, and (D) for ethylene glycol. Other conditions: enzyme load: 3% w/w; solvent: tert-butanol for ethylene glycol. Other conditions: enzyme load: 3% w/w; solvent: tert-butanol (30% v/v); closed (30% stirred batch reactor; agitation speed: 400 (30%v/v); v/v);closed closed stirred batchspeed: reactor;400 agitation speed: 400rpm; rpm;molar molar ratioEC:Gly EC:Gly1:3. 1:3. stirred batch reactor; agitation rpm; molar ratio EC:Gly 1:3. ratio

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4. Conclusions Glycerol carbonate can be synthesized from glycerol and organic carbonates like dimethyl carbonate and ethylene carbonate employing enzymatic processes. In fact, the latter reaction is presented for the first time here using an enzymatic transesterification reaction. The two reactions are first conducted with different free and immobilized enzymes and their performance was compared. For the transesterification with dimethyl carbonate, CALB was the free enzyme of choice, whereas Eversa Transform 2.0 performed better for the reaction with ethylene carbonate, a reaction, that in the end, showed a much better performance. For this reason, subsequent studies with this reaction and enzyme optimized the operation conditions using tert-butanol as solvent in a closed system to minimize possible evaporation, a concentration of enzyme Eversa Transform 2.0 of 3% w/w, a molar excess of EC:Gly from 3:1 to 9:1 and a temperature from 50 to 70 ◦ C. The evolution of the concentration of the components involved was determined by ion exclusion HPLC, while a kinetic model was proposed to fit data retrieved in runs performed in a closed reactor at several molar ratios (runs 1 to 3) and at several temperatures (runs 1, 4, and 5). The model proposed and validated included a transesterification reaction taking place in parallel to a polymerization-depolymerization reaction affecting EC. The first one was very influenced by temperature, as its reaction rate rocketed between 60 and 70 ◦ C. Author Contributions: M.L. and J.E. conceived the research; M.L. designed the experiments; A G.-L.; D.V. and D.E.B. performed the experiments and analyzed the samples; J.E. developed the original analytical procedures, while A.G.-L. and D.V. developed a modified analytical HPLC procedure for this reacting system and analyzed the retrieved data, including the preliminary statistical analysis; J.E., P.Y. and M.L. performed the final statistical analysis and wrote the paper. All the authors read and approved the final manuscript. Funding: This work has been supported by MINECO (Ministerio de Economía y Competitividad, Spain) under contracts CTQ2013-45970-C2-1-R and PCIN-2013-021-C02-01 and by the Complutense University through BSCH-UCM, GR35/10-A 910134. Acknowledgments: The kind gift of Lipozyme 435, Lipozyme TL IM, Lipozyme TL 100L, Lipozyme CALB L, and Eversa Transform 2.0 by Ramiro Martinez (Novozymes Spain, Pozuelo de Alarcon-Madrid-Spain) is gratefully acknowledged. This work was funded by MICINN under contracts CTQ-2013-45970-C2-1-R and PCIN-2013-021-C02-01. Conflicts of Interest: The authors declare no conflict of interest. The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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17. 18.

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