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Transesterification of triolein and methanol with Novozym 435 using co-solvents
Fuel 263 (2020) 116600
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Full Length Article
Transesterification of triolein and methanol with Novozym 435 using cosolvents
T
⁎
Hidetoshi Kuramochia, , Zhenyi Zhanga, Kazuko Yuia, Takuro Kobayashia, Kouji Maedab a b
Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Department of Chemical Engineering, University of Hyogo, 2167 Himeji, Hyogo 671-2201, Japan
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Co-solvent effect Enzymatic methanolysis Liquid-liquid equilibrium Transfer free energy LLE-UNIFAC model
To enhance the reaction rate and yield during biodiesel synthesis with Novozym 435 (an immobilized lipase), a co-solvent method was applied, after which the relationships between reaction time and reaction yield during the single-phase enzymatic transesterification of triolein and methanol in the presence of co-solvents such as tetrahydrofuran, acetone, and hexane were investigated. The addition of hexane led to inactivation of Novozym 435, whereas in the presence of acetone or tetrahydrofuran, the reaction was accelerated and the yield was higher than with a conventional solvent-free enzymatic reaction. There was a clear difference in the dispersion of Novozym 435 particles between with and without co-solvents. The co-solvents prevented particle aggregation from occurring during the reaction without co-solvent. This is the likely reason for the enhanced reaction yield in the presence of co-solvents. To better understand the difference among the co-solvents employed, we estimated phase compositions during transesterification using the LLE (liquid-liquid equilibrium)-UNIFAC (UNIversal quasichemical Functional group Activity Coefficients) model, and examined the effect of these compositions on the reaction and dispersion of the glycerin by-product phase. Using the model, we also accounted for why hexane led to inactivation by estimating the transfer free energy of methanol, glycerin, and water between the initial bulk reaction solution and the water layer on the enzyme surface.
1. Introduction Biodiesel, consisting of fatty acid methyl esters (FAMEs), is now produced in quantity from a wide variety of vegetable oils and used
⁎
cooking oils; its global production in 2017 was 41 billion liters [1]. The homogeneous alkali catalyst method is normally employed for biodiesel synthesis (transesterification of triglycerides [TGs] with methanol), but this requires excess methanol to enhance the reaction yield as well as
Corresponding author. E-mail address: [email protected] (H. Kuramochi).
https://doi.org/10.1016/j.fuel.2019.116600 Received 15 March 2019; Received in revised form 15 October 2019; Accepted 5 November 2019 Available online 09 December 2019 0016-2361/ © 2019 Elsevier Ltd. All rights reserved.
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and hexane (96%), used as co-solvents, were purchased from Wako pure Chemical Industries, Ltd. (Tokyo, Japan). To determine the ester yield, the following standard reagents purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) were used: triolein (≥99%), methyl oleate (≥99%), monoolein (99%), and diolein mixture of 1,3- and 1,2-isomers (99%). Acetonitrile (99.8%), isopropyl alcohol (99.7%), and hexane (96%), purchased from Wako Pure Chemical Industries, were used as organic solvents of the mobile phase in the high-performance liquid chromatography (HPLC) assays. Pure water (≤18.2 MΩ·cm), used as the mobile phase in the HPLC assay, was supplied by a Synthesis A10 water purification system followed by an Elix 10 water purification system (Millipore Corp., Billerica, MA, USA).
hot-water washing of the crude biodiesel to purify it. Recent studies have shown that the use of heterogeneous catalysts, such as metal oxides [2], ion-exchange resins [3,4] and immobilized lipases [5,6] can overcome these disadvantages. Solvent-free transesterification using a commercial immobilized lipase such as Novozym 435 (N435) can significantly reduce the amount of methanol required [5,6]. For biodiesel synthesis by N435, however, a single-phase reaction solution is needed at the beginning of the reaction, because phase-separated methanol inactivates N435. In order to add methanol up to the reaction equivalent, it must be added in two or three steps to prevent phase separation of the methanol. The most common form of biodiesel production using homogeneous alkali catalysts has two phases (methanol-rich and TGrich phases); and the addition of ethers such as tetrahydrofuran (THF) and liquefied dimethyl ether (DME) results in single-phase transesterification, thus significantly increasing the reaction rate [7,8]. If such cosolvents could be used in enzymatic biodiesel production, single-step addition of methanol up to the reaction equivalent would be possible, and the reaction would proceed much faster. In earlier works [9,10], a faster rate was observed in a transesterification system of TGs and methanol with N435 in the presence of those ethers. The effects of organic solvents except for alcohols (a substrate) on enzyme-catalyzed transesterification of vegetable oils have also been reported in earlier studies [10–13]. Unfortunately, the experimental conditions in those papers except for one paper [13], which were almost solvent-rich, differ significantly from those of actual biodiesel synthesis. The actual synthesis should use the amount of co-solvents to be minimized for a single-phase transesterification, because the co-solvents must be recovered with a distillation after the transesterification. Talukder et al. [13] reported the effect of mixing ratio of co-solvent to oil on reaction yield. However, the amount of co-solvents required to be added for the single-phase formation of a reaction system and the reaction yield in such a single-phase system are unknown. Moreover, although aggregation of N435 occurs during biodiesel synthesis due to the adsorption of glycerin to the surface of the enzyme-resins and is also affected by what the reaction vial is made of [14], the effect of co-solvent on the aggregation and glycerin phase has hitherto not been investigated. In this study, the transesterification of triolein and methanol in the presence of several selected co-solvents such as THF, acetone, and hexane, was performed using N435. The relationships between the reaction time and reaction yield during single-phase transesterification were examined. Simultaneously, dispersion of N435 and glycerin byproduct was observed, and the effects of co-solvents were investigated and contrasted with our observations of dispersion in previous studies on transesterification without co-solvents [14]. To better understand the effects of co-solvents on the dispersion and the reaction performance, in addition, phase equilibrium composition during the reaction progress was estimated using the LLE-UNIFAC model. We also attempted to calculate the potential for the transfer of water, glycerin, and methanol between the initial bulk solution and the water layer on the enzyme surface, and, using the calculated results, to investigate the reason for the inactivation caused by addition of hexane.
2.2. Experimental procedure In our previous study on an enzymatic transesterification of triolein and methanol [14], a conventional stepwise batch transesterification (two-step addition of methanol up to the reaction equivalent), as reported by Watanabe et al. [6], was employed. In the presence of cosolvents, the two-step addition of methanol was changed to one-step addition. The experimental procedure is, briefly, as follows. First, 0.4 g of N435 was placed in a 50-mL glass vial, followed by 9.65 g of triolein and an aliquot of co-solvent. In this study, tetrahydrofuran (THF), acetone, and hexane were selected as the co-solvents, because Talukder et al. [13] reported that THF and acetone were active co-solvents, whereas hexane was not. However, their experimental conditions as well as research objective differed from ours. We focus on minimizing the amount of co-solvents, which must be removed by distillation after the transesterification, and also their effect on N435 aggregation. Therefore, we examined the amount of each co-solvent required to keep the initial reaction system homogeneous. As a result, the amount of THF, acetone, and hexane added into the reaction system were 1.64, 3.33, and 5.6 g, respectively. Finally, methanol was slowly loaded, ensuring no direct contact with the N435. The molar ratio of methanol to triolein was three, namely the reaction equivalent, according to a report by Watanabe et al. [6]. The ratio is considered to be sufficient for the reaction, because we also observed a high yield of 93.2% in our previous work [14]. The weight fractions of all components at the beginning of the reaction are summarized in Table S1. The vial was immersed in a thermostatically-controlled water bath at 30 °C, and shaken at 130 rpm. At appropriate intervals, e.g., 2, 4, 11 h, etc., 100 µL of the reaction solution was sampled with a micro syringe. The sample was then diluted with isopropanol and the concentrations of triolein, diolein, monoolein, and methyl oleate in the sample were determined using a HPLC-UV system with a 3.0 × 150 mm ODS column as described in our previous study on the transesterification of triolein with a homogeneous alkali catalyst [8]. In addition, the concentrations were converted to the mole fraction (x) of methyl oleate (ME), monoolein (MO), diolein (DO), and triolein (TO). The yield of methyl oleate (YME) was calculated by the mole fraction and stoichiometric factor of each compound in the transesterification of TO, DO, and MO as follows.
YME =
2. Experimental section
xME 3xTO+2xDO+xMO+xME
(1)
2.1. Materials 3. Calculations by UNIFAC model Novozym 435 (N435) was purchased from Novozymes Japan Ltd. (Chiba, Japan). The enzyme, from Candida antarctica, was immobilized on particles of acrylic resin. The supplier reports its activity to be 10,000 U/g. Triolein (technical grade) and methanol (99.7%) used in the transesterification process were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), respectively, and were used without further purification. A preliminary analytical test showed the oleic acid content of the triolein to be 0.564 wt%. Tetrahydrofuran (99.8%), acetone (99.5%),
The UNIFAC (UNIversal quasichemical Functional group Activity Coefficients) model is a group contribution method based on molecular thermodynamics [15], that is widely used to predict nonideality, namely the activity coefficient, in nonelectrolyte mixtures. The model describes the activity coefficient γi of component i by a combination of the combinatorial contribution due to differences in molecular size and shape, and the residual contribution due to differences in intermolecular interaction: 2
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Table 1 UNIFAC group assignment in this study. Compound
UNIFAC group assignment
Triolein Methyl oleate Glycerin Methanol THF Acetone Hexane
3 2 1 1 3 1 2
× × × × × × ×
CH3, 41 × CH2, 1 × CH, 3 × CH]CH, 3 × CH2COO CH3, 13 × CH2, 1 × CH]CH, CH2COO CH, 2 × CH2, 3 × OH CH3, 1 × OH CH2, 1 × FCH2O CH3, 1 × CH3CO CH3, 4 × CH2
lnγi = lnγiComb. + lnγiRes.
(2)
Many UNIFAC models have been reported since 1975. In our previous works [16,17], we reported the LLE-UNIFAC model [18] to be useful in terms of predicting the liquid-liquid equilibrium (LLE) in the biodiesel production process. Therefore, it was employed in the present study. In the UNIFAC model, the molecules in a system are divided into several groups, as shown in Table 1, after which the activity coefficient γi of component i is calculated according to the interaction between the groups and their size parameters. The size and interaction parameter values were cited from the LLE-UNIFAC parameter tables [18]. If the activity coefficient is calculated from the UNIFAC model, LLE can be obtained by solving the following mass balance and equilibrium equations derived from the criterion for phase equilibrium that the fugacity of component i in one phase is equal to that in the other phase: Fig. 1. Schematic illustration of inactivation of enzyme by methanol (MeOH) and glycerin (Gly), and three types of transfer free energy (ΔGtr) of MeOH, Gly, and water (W) between initial bulk solution (IBS) and the water layer on the enzyme surface (WL).
N
1=
∑ x iJ
(3)
i= 1
For the criterion for LLE,
γiI·x iI = γiII·x iII
ΔGi,tr B→A = μiA − μiB = μiA, * − μiB,*
(4)
B, ∞
γ = μio + RTlnγiA, ∞ − (μio + RTlnγiB,∞) = −RTln ⎜⎛ iA, ∞ ⎞⎟ γ ⎠ ⎝ i
where x refers to the mole fractions of component i. Superscripts I and II denote the liquid phase in a two-liquid system. For the LLE calculation, xiI and xiII were predicted by solving Eqs. (2), (3), and (4) and an additional constraint condition, which is an overall mass balance equation (the lever rule and feed composition) using the Newton-Raphson technique. As described in Section 4.2, we evaluated the equilibrium compositions of the oil phase and the glycerin phase that emerged during the transesterification reaction. To evaluate the variation of the phase compositions during the progress of the transesterification reaction, we first calculated the mole amounts of reactants and products such as TO, methanol, ME, and glycerin at a given methyl oleate yield (YME) and then, with the mole amount of co-solvent (acetone or THF), two phase LLE between glycerin phase and oil phase were calculated. Here, we assumed that N435 did not affect the phase equilibria and neglected the presence of esters other than ME. The mole fractions in the oil phase and glycerin phase, xiOil and xiGly, respectively were determined using Eqs (2)–(4), and the amount and equilibrium compositions of the two phases were estimated. Moreover, the transfer free energy of component i from B and A (ΔGtr i,B→A) was calculated as follows: A
The superscript * denotes a standard state (at infinite dilution). ΔGtr i,B→A can be calculated from the activity coefficients in solvents A and B estimated using the UNIFAC model. If a co-solvent leads to inactivation with several hours after the beginning of transesterification, the structure of the enzyme may be affected; e.g., as shown in Fig. 1a, inactivation is caused by increased methanol concentration and decreased water content on the surface of the enzyme, as reported by Fu and Vasudevan [10]. In addition, another inactivation due to adsorption of glycerin on the enzyme [20,21] may be promoted as shown in Fig. 1a. As shown in Fig. 1b–d, therefore, we evaluated the potential of this inactivation using the following three parameters: ΔGtr MeOH,IBS→WL: transfer free energy of methanol (MeOH) in the initial bulk solution (IBS) to the water layer on the enzyme surface (WL), ΔGtr Gly,IBS→WL: transfer free energy of glycerin (Gly) in the initial bulk solution (IBS) to the water layer on the enzyme surface (WL), and Δ Gtr W,WL→IBS: transfer free energy of water (W) from WL to IBS. Both parameters are given by: IBS
IBS
⎛ xMeOH ·γMeOH ⎞ tr ΔGMeOH,IBS → WL = − RTln ⎜ WL WL ⎟ ⎝ xMeOH ·γMeOH ⎠
(7)
B
tr A B o A o B ΔGi,B → A = μ i − μ i = μ i + RTlnx i γi − (μ i + RTlnx i γi )
tr ΔGGly, IBS → WL = − RTln
B
B ⎛ x i γi ⎞ = −RTln ⎜ x AγA ⎟ ⎝ i i ⎠
(6)
(5)
IBS IBS ⎛ x Gly ·γGly ⎞ ⎜ x WL ·γ WL ⎟ ⎝ Gly Gly ⎠
WL WL ⎛ x W ·γW ⎞ tr ΔGW, WL → IBS = − RTln ⎜ IBS IBS ⎟ ⎝ x W ·γW ⎠
where μi refers to the chemical potential of component i in each phase. The superscript o denotes a standard state (pure liquid of component i). If the conentration can be assumed to be at infinite dilution, it can be calculated from its infinite dilution activity coefficients (γi∞) in both phases [19]:
(8)
(9)
In the calculation of activity coefficient, we assumed that methanol or glycerin in WL and glycerin or water in IBS are in infinite dilution. Furthermore, WL is defined as pure water (xWWL = 1 and γWWL = 1). 3
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than those in the absence of co-solvent [14], namely under conventional solvent-free conditions. In contrast, YME values in the case of either THF or acetone were much higher than those under conventional conditions. Although there was no difference in the YME value at 2 h between hexane and acetone, inactivation was clearly observed after 4 h in the case of hexane. This indicates that not only methanol but also glycerin may be responsible for this inactivation. The reason for the inactivation is discussed in Section 4.3. With the addition of THF or acetone, YME values at 74 h exceeded 0.80, while YME at the same time under the conventional conditions reached only 0.623. In terms of reaction activity, THF and acetone were active co-solvents, while hexane was a negative co-solvent. This was the same as reported by Talukder et al. [13]. The active co-solvents significantly enhanced the reaction rate over 11 h. For example, the difference in reaction time to achieve the yield of 0.6 between with and without active co-solvent was over 2 days. We therefore examined the dispersion of N435 particles under each condition. Fig. 3a–c shows photographs of the particles during transesterification of triolein from 2 to 24 h. In the absence of co-solvent [14], no clear aggregation of the particles was observed up to 2 h. After 2 h, bulky and thin-layer aggregates were observed: the former aggregates grew with increasing reaction time. We speculated that the glycerin by-product acted as a form of adhesive in the interstices of the immobilized enzyme particles. In the presence of THF as well as acetone, no such bulky aggregates were observed. These results demonstrate that co-solvents play a role in preventing the aggregation of enzyme particles. In other words, the surface area of N435 was kept almost constant by the addition of co-solvent. This is likely the reason for the better reaction yield and rate in the presence of active co-solvents. The effect is very advantageous in terms of reaction rate and yield, because our previous work showed the bulky aggregates to have a very low reaction activity [14]. From 11 to 24 h, in the case of THF, however, small rigid or gel-like glycerin droplets coated with several particles, rather than aggregates of N435, were clearly observed, as shown in Fig. 3a. On the other hand, there were soft glycerin phases that were able to grow in the presence of acetone. From an industrial application perspective, acetone is better, since the glycerin phase was completely separated from the methyl oleate phase and N435 particles in the presence of acetone, as shown in Fig. 3b. This conclusion is different from that of an earlier work on the co-solvent effect of THF and acetone [13], because it focused on only reaction yield. Our previous study [14] showed the material of which the reaction vial was made strongly affected the aggregation of N435 and the reaction yield. Indeed, the use of a polypropylene vial produced only bulky aggregate (clumps of densely packed particles) and a reaction yield of around 0.45. In this study, therefore, the same transesterification in the presence of THF and acetone was performed using a 50-mL polypropylene vial. The results are shown in Fig. 4 and Table 3. The reaction curves were almost the same as those using a glass vial, showing that, in the presence of active co-solvents, the material of which the vial was made had little effect on the reaction. This can remove a negative factor from the reaction and is also an advantage from an industrial application perspective. Our observations on the effects of co-solvents on reaction yield significantly differed from those in earlier works [10–12], where immobilized lipases were used under solvent-rich conditions. Under solvent-rich conditions, hexane proved an effective solvent for enzymecatalyzed transesterification. In contrast, though, THF inactivated the activity of the enzyme, revealing that the solvent composition in the reaction system has a strong effect on the activity of lipase in N435. It should be noted that lipase activity under solvent-rich conditions cannot be used for predicting activity under solvent-poor conditions, such as those employed in the present study. The reason for the inactivation in the case of hexane, which is contrary to the earlier works, will be discussed in Section 4.3 using a thermodynamic approach.
Fig. 2. Reaction yield (YME) versus reaction time (t) in the enzyme-catalyzed transesterification of triolein in the presence of co-solvents (hexane, tetrahydrofuran (THF), and acetone) using a 50-mL glass vial at 30 °C. *: our previous work [14], (gv): glass vial.
From these assumptions, Eqs. (7)–(9) are transformed: IBS
IBS
⎛ xMeOH ·γMeOH ⎞ tr ΔGMeOH,IBS → WL = − RTln ⎜ ⎟ WL, ∞ γMeOH ⎝ ⎠
(10)
IBS,∞
tr ΔGGly, IBS → WL = − RTln
⎛ γGly ⎞ ⎜ γ WL, ∞ ⎟ ⎝ Gly ⎠
(11)
⎛ 1 ⎞ tr ΔGW,WL → IBS = − RTln ⎜ IBS, ∞ ⎟ ⎝ γW ⎠
(12)
where superscript ∞ refers to infinite dilution. The effect of co-solvents on WL is discussed based on these parameters. 4. Results and discussion 4.1. Effect of co-solvents on the reaction yield and immobilized enzyme particles The reaction yields of methyl oleate (YME) with or without co-solvents are plotted as a function of the reaction time (t) as shown in Fig. 2 and Table 2. YME values in the presence of hexane were much lower Table 2 Reaction yield (YME) with and without co-solvents (hexane, tetrahydrofuran (THF), acetone) during methanolysis of triolein by Novozym 435 in a glass vial. t/h
YME with hexane/–
YME with THF/–
YME with acetone/–
YME without cosolvent*/–
2 4 11 24 48 64 72 74 94 96
0.044 0.067 n.m. 0.081 n.m. n.m. n.m. n.m. n.m. n.m.
n.m. 0.266 0.381 0.681 0.778 n.m. 0.826 n.m. n.m. n.m.
0.049 0.109 0.375 0.603 0.768 0.835 0.832 n.m. n.m. 0.846
0.136 0.197 0.344 0.438 0.516 n.m. n.m. 0.628 0.685 nm
*Our previous work according to a conventional solvent-free system [14], n.m.: not measured. 4
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Fig. 3. Novozym 435 particles during the transesterification of triolein with and without co-solvent using a 50-mL glass vial. *: our previous work [14].
Table 3 Reaction yield (YME) with and without co-solvents (hexane, tetrahydrofuran (THF), acetone) during methanolysis of triolein by Novozym 435 in a polypropylene vial. t/h
YME with THF/–
YME with acetone/–
YME without co-solvent*/–
2 4 11 24 46 64 72 74 94 96
n.m. 0.255 0.459 0.725 0.872 n.m. 0.893 n.m. 0.897 n.m.
0.042 0.106 0.354 0.608 0.760 0.815 0.831 n.m. n.m. 0.843
0.142 0.190 0.306 0.373 0.421 n.m. n.m. 0.447 0.454 n.m.
*Our previous work according to a conventional solvent-free system [14], n.m.: not measured.
estimated using the LLE-UNIFAC model [18]. Here, we assumed that there were only two phases, oil phase and glycerin phase, and neglected the effect of N435 on the LLE. The detailed calculation method was already described in the Section 3. Methanol and co-solvents were assumed to dissolve in either or both of the oil phase and the glycerin phase, and there was no third phase. The temperature was set to 30 °C and the initial amounts of triolein, methanol, and co-solvents were the same as those provided in Section 2. For four fixed methyl oleate yields (YME) from 0.3 to 0.9, the equilibrium compositions were determined. The calculated results for the oil and glycerin phases are shown in Fig. 5a, b. For the oil phase, the concentrations of reactants such as methanol and triolein in the case of THF exceeded those in the case of acetone. The addition of acetone led to more diluted concentrations of both reactants. The reaction rate in the presence of THF therefore
Fig. 4. Reaction yield (YME) versus reaction time (t) in the enzyme-catalyzed transesterification of triolein in the presence of co-solvents (hexane, tetrahydrofuran (THF), and acetone) using a 50-mL polypropylene vial at 30 °C. *: our previous work [14], (pv): polypropylene vial.
4.2. Estimation of the phase equilibrium of the reaction system Although both THF and acetone were effective co-solvents for the transesterification of triolein, THF was superior in terms of enhancing the reaction rate, as shown in Figs. 2 and 4. Meanwhile, acetone was superior to THF with respect to separation of the glycerin by-product, as shown in Fig. 3a, b. To better understand these differences, variation in phase equilibrium composition during the transesterification with co-solvent was 5
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Fig. 5. Estimated compositions of oil phase in equilibrium with glycerin phase with THF or acetone as a cosolvent, during the transesterification of triolein calculated using the LLE-UNIFAC model [18]. a) Mole fraction of component i in the oil phase (xiOil) and glycerin phase (xiGly) at reaction yields (YME) of 0.3, 0.5, 0.7 and 0.9, in the presence of THF as a co-solvent. b) Mole fraction of component i in the oil phase (xiOil) and glycerin phase (xiGly), at reaction yields (YME) of 0.3, 0.5, 0.7 and 0.9, in the presence of acetone as a co-solvent.
to cause the methanol-driven inactivation and/or the glycerin-driven inactivation. Table 4 shows the transfer free energies of methanol and glycerin from the initial bulk solution to the water layer on the enzyme tr surface (ΔGtr MeOH,IBS→WL and ΔGGly,IBS→WL, respectively) and the activity coefficients used for calculating the energy for all the reaction systems. In the calculated results of ΔGtr MeOH,IBS→WL, THF and hexane have lower values. This suggests that both solvents have higher potentials of the methanol-driven inactivation than acetone and non-co-solvent condition. Furthermore, the results of ΔGtr Gly,IBS→WL indicate that it is easier to bring about the glycerin-driven inactivation in the presence of hexane. Therefore, we considered that the addition of hexane has a larger negative impact on the enzymatic transesterification in terms of methanol- and glycerin-driven inactivations than the other co-solvents. Although the ΔGtr Gly,IBS→WL value for hexane is equal to that for the nonco-solvent condition, the reaction rate without hexane is expected to be much faster than that with hexane because the calculated activities of TO and MeOH in the absence of hexane are much higher as shown in Table 4. The larger amounts of products such as ME and glycerin due to the high reaction rate might mitigate the two inactivations. Indeed, the conventional two-step addition of methanol [6] demonstrates that an increase of ME concentration is able to dissolve more methanol in bulk solution. This approach using the UNIFAC model may thus be useful for evaluating co-solvent effects. We also estimated the possibility that cosolvents destabilize the water layer. The transfer free energy of water
appears to be faster than that with acetone as shown in Fig. 4. The content of co-solvents in the glycerin phase differed significantly, with the concentration of acetone being much higher than that of THF. Since acetone is a less viscous liquid, the high concentration of acetone resulted in a soft glycerin phase. Reducing the viscosity of the glycerin phase was useful for removing viscous glycerin from the reaction system. In contrast, the concentration of THF in the glycerin phase was much lower, indicating that the glycerin phase was more viscous. This is a fairly good explanation for the formation of the small rigid glycerin phases coated with N435 particles shown in Fig. 3b, a. If the removal of the glycerin phase is the key factor in biodiesel production with N435, acetone is superior to THF as a co-solvent. 4.3. Estimation of transfer free energy of methanol and water It is well known that contact with phase-separated methanol inactivates the immobilized lipase. It is thought that hydrophilic methanol molecules remove the water molecules on the surface of the lipase [10]. Moreover, adsorption of glycerin on the lipase serves as a barrier to transport reactants resulting in a kind of inactivation of the lipase [20,21]. Since the inactivation clearly occurred after 4 h in our experiments using hexane, the effect of the produced glycerin should also be considered in addition to the methanol-driven inactivation. Therefore, we tried to investigate whether the addition of hexane tends 6
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potentially useful for evaluating the effects of co-solvents on enzymatic transesterification.
Table 4 Activity coefficients of methanol (MeOH), glycerin (Gly) and water (W) in the initial bulk solution (IBS) and the water layer on the enzyme surface (WL) estimated by LLE-UNIFAC model [18], mole fraction of MeOH in ISB, activity of MeOH and TO in ISB, and transfer free energy of MeOH, Gly, and W between IBS and WL calculated by Eqs. (10)–(12).
γMeOHIBS/– γGLyIBS, ∞/– γMeOHWL, ∞/– γGLyWL, ∞/– γWIBS, ∞/– xMeOHIBS/– aMeOHIBS/– aTOIBS/– −1 ΔGtr MeOH,IBS→WL/kJ mol −1 ΔGtr Gly,IBS→WL/J mol −1 ΔGtr W,WL→IBS/kJ mol
With THF
With acetone
With hexane
No cosolvent
1.98 91.2 2.24 0.583 10.7 0.492 0.973 0.0743 2.10 −12.7 5.97
1.92 49.0 2.24 0.583 9.73 0.324 0.622 0.116 3.22 −11.2 5.73
3.04 271 2.24 0.583 29.2 0.302 0.917 0.0137 2.25 −15.5 8.50
1.60 275 2.24 0.583 17.2 0.500 0.801 0.467 2.58 −15.5 7.16
Acknowledgement This work was supported by JSPS (the Japan Society for the Promotion of Science) KAKENHI Grant Number 21360449. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fuel.2019.116600. References [1] R REN21, Renewables 2018 GLOBAL STATUS REPORT, http://www.ren21.net/wpcontent/uploads/2018/06/178652_GSR2018_FullReport_web_-1.pdf [accessed October 1, 2018]. [2] Kouzu M, Hidaka J. Transesterification of vegetable oil into biodiesel catalyzed by CaO: a review. Fuel 2012;93:1–12. [3] Shibasaki-Kitakawa N, Honda H, Kuribayashi H, Toda T, Fukumura T, Yonemoto T. Biodiesel production using anion ion-exchange resin as heterogeneous catalyst. Bioresour Technol 2007;98:416–21. [4] Shibasaki-Kitakawa N, Tsuji T, Chida K, Kubo M, Yonemoto T. Simple continuous production process of biodiesel fuel from oil with high content of free fatty acid using ion-exchange resin catalysts. Energy Fuels 2010;24:3634–8. [5] Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, et al. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. JAOCS 1999;76(7):789–93. [6] Watanabe Y, Shimada Y, Sugihara A, Noda H, Fukuda H, Tominaga Y. Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase. JAOCS 2000;77(4):355–60. [7] Boocock DGB, Konar SK, Mao V, Sidi H. Fast one-phase oil-rich processes for the preparation of vegetable oil methyl esters. Biomass Bioenergy 1996;11:43–50. [8] Kuramochi H, Maeda K, Osako M, Nakamura K, Sakai S. Superfast transesterification of triolein using dimethyl ether and a method for high-yield transesterification. Ind Eng Chem Res 2008;47:10076–9. [9] Maeda K, Kuramochi H, Arafune K, Itoh K, Yamamoto T. Transesterification of triolein and methanol by Novozym 435 with dimethyl ether. J Chem Eng Jpn 2017;50(12):924–8. [10] Fu B, Vasudevan PT. Effect of organic solvents on enzyme-catalyzed synthesis of biodiesel. Energy Fuels 2009;23:4105–11. [11] Gog A, Roman M, Toşa M, Paizs C, Irimie FD. Biodiesel production using enzymatic transesterification – Current state and perspectives. Renewable Energy 2012;39:10–6. [12] Guldhe A, Singh B, Mutanda, Permaul K, Bux F. Advances in synthesis of biodiesel via enzyme catalysis: novel and sustainable approaches. Renew Sustain Energy Rev 2015;41:1447–64. [13] Talukder MMR, Puah SM, Wu JC, Won CJ, Chow Y. Lipase-catalyzed methanolysis of palm oil in presence and absence of organic solvent for production of biodiesel. Biocatal Biotransform 2006;24(3):257–62. [14] Kuramochi H, Kobayashi T, Maeda K. Aggregation of immobilized enzyme during transesterification of triolein and methanol, and the effect of two types of aggregates on reaction yield. Fuel 2020;260:116343. [15] Fredenslund A, Jones RL, Prausnitz JM. Group-contribution estimation of activity coefficients in non-ideal liquid mixtures. AIChE J 1975;21:1086–99. [16] Kuramochi H, Maeda K, Kato S, Osako M, Nakamura K, Sakai S. Application of UNIFAC models for prediction of vapor-liquid and liquid-liquid equilibria relevant to separation and purification processes of crude biodiesel. Fuel 2009;8:1472–7. [17] Maeda K, Kuramochi H, Asakuma Y, Fukui K, Tsuji T, Osako M, et al. De-emulsification of biodiesel mixtures with dimethyl ether. Chem Eng J 2011;169:226–30. [18] Magnussen T, Rasmussen P, Fredenslund A. UNIFAC parameter table for prediction of liquid-liquid equilibria. Ind Eng Chem Process Des Dev 1981;20:331–9. [19] Kobayashi T, Kuramochi H, Xu K-Q, Maeda K. Simple solvatochromic spectroscopic quantification of long-chain fatty acids for biological toxicity assay in biogas plants. Environ Sci Pollut Res [in press]. [20] Dossat V, Combes D, Marty A. Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production. Enzyme Microb Technol 1999;25:194–200. [21] Xu Y, Nordblad M, Nielsen MP, Brask J, Woodley JM. In situ visualization and effect of glycerol in lipase-catalyzed ethanolysis of rapeseed oil. J Mol Catal B Enzym 2011;72:213–9.
γMeOHIBS: activity coefficient of MeOH in IBS, γGlyIBS, ∞: infinite dilution activity coefficient of Gly in IBS, γMeOHWL, ∞: infinite dilution activity coefficient of MeOH in WL, γGlyWL, ∞: infinite dilution activity coefficient of Gly in WL, γWIBS, ∞ : infinite dilution activity coefficient of W in IBS, xMeOHIBS: mole fraction of MeOH in ISB, aMeOHIBS: activity of MeOH in ISB, aTOIBS: activity of TO in tr ISB,ΔGtr MeOH,IBS→WL: transfer free energy of MeOH from IBS to WL, GGly,IBS→WL: transfer free energy of Gly from IBS to WL,ΔGtr W,WL→IBS: transfer free energy of W from WL to IBS.
from the water layer to the bulk solution (ΔGtr WL,W→IBS) was calculated. The results are shown in Table 4. All the values are high positive values, meaning that water molecules on the enzyme surface are very unlikely to transfer to the initial bulk solution. The present co-solvents are considered to have little effect on the water layer if the transfer of methanol is ignored. 5. Conclusions We investigated the effects of three co-solvents on the enzyme-catalyzed transesterification of triolein and methanol. Hexane led to inactivation of Novozym 435. However, the reaction yield in the presence of acetone or tetrahydrofuran (THF) was found to be much higher than without co-solvent. The results were almost the same as reported in an earlier work. However, we first revealed the other effects due to the cosolvents as follows: both active co-solvents inhibited the aggregation of Novozym 435 particles that occurred in the absence of the co-solvents, resulting in enhanced reaction rate and yield. In addition, there was a clear difference in the formation and dispersion of the glycerin phase between THF and acetone: viscous and rigid glycerin droplets coated with Novozym 435 particles in the case of THF, and soft glycerin phases, which can form a single phase, in the case of acetone. Therefore, acetone is useful for removing the glycerin phase from the reaction system. The phase equilibrium composition in the reaction system with co-solvents, estimated using the LLE-UNIFAC model, indicated that acetone significantly dilutes the glycerin phase. Finally, the transfer free energy of methanol, glycerin, and water between the initial bulk solution and water layer on the enzyme surface was estimated using the LLE-UNIFAC model. The lower transfer energies of methanol and glycerin from the bulk to the water layer in the presence of hexane indicate that hexane has a higher potential of the methanol-driven and glycerin-driven inactivations. This may be the reason why hexane led to a lower reaction yield. Analysis based on the LLE-UNIFAC model is thus
7
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Full Length Article
Transesterification of triolein and methanol with Novozym 435 using cosolvents
T
⁎
Hidetoshi Kuramochia, , Zhenyi Zhanga, Kazuko Yuia, Takuro Kobayashia, Kouji Maedab a b
Center for Material Cycles and Waste Management Research, National Institute for Environmental Studies, 16-2 Onogawa, Tsukuba, Ibaraki 305-8506, Japan Department of Chemical Engineering, University of Hyogo, 2167 Himeji, Hyogo 671-2201, Japan
G R A P H I C A L A B S T R A C T
A R T I C LE I N FO
A B S T R A C T
Keywords: Co-solvent effect Enzymatic methanolysis Liquid-liquid equilibrium Transfer free energy LLE-UNIFAC model
To enhance the reaction rate and yield during biodiesel synthesis with Novozym 435 (an immobilized lipase), a co-solvent method was applied, after which the relationships between reaction time and reaction yield during the single-phase enzymatic transesterification of triolein and methanol in the presence of co-solvents such as tetrahydrofuran, acetone, and hexane were investigated. The addition of hexane led to inactivation of Novozym 435, whereas in the presence of acetone or tetrahydrofuran, the reaction was accelerated and the yield was higher than with a conventional solvent-free enzymatic reaction. There was a clear difference in the dispersion of Novozym 435 particles between with and without co-solvents. The co-solvents prevented particle aggregation from occurring during the reaction without co-solvent. This is the likely reason for the enhanced reaction yield in the presence of co-solvents. To better understand the difference among the co-solvents employed, we estimated phase compositions during transesterification using the LLE (liquid-liquid equilibrium)-UNIFAC (UNIversal quasichemical Functional group Activity Coefficients) model, and examined the effect of these compositions on the reaction and dispersion of the glycerin by-product phase. Using the model, we also accounted for why hexane led to inactivation by estimating the transfer free energy of methanol, glycerin, and water between the initial bulk reaction solution and the water layer on the enzyme surface.
1. Introduction Biodiesel, consisting of fatty acid methyl esters (FAMEs), is now produced in quantity from a wide variety of vegetable oils and used
⁎
cooking oils; its global production in 2017 was 41 billion liters [1]. The homogeneous alkali catalyst method is normally employed for biodiesel synthesis (transesterification of triglycerides [TGs] with methanol), but this requires excess methanol to enhance the reaction yield as well as
Corresponding author. E-mail address: [email protected] (H. Kuramochi).
https://doi.org/10.1016/j.fuel.2019.116600 Received 15 March 2019; Received in revised form 15 October 2019; Accepted 5 November 2019 Available online 09 December 2019 0016-2361/ © 2019 Elsevier Ltd. All rights reserved.
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and hexane (96%), used as co-solvents, were purchased from Wako pure Chemical Industries, Ltd. (Tokyo, Japan). To determine the ester yield, the following standard reagents purchased from Sigma-Aldrich Inc. (St. Louis, MO, USA) were used: triolein (≥99%), methyl oleate (≥99%), monoolein (99%), and diolein mixture of 1,3- and 1,2-isomers (99%). Acetonitrile (99.8%), isopropyl alcohol (99.7%), and hexane (96%), purchased from Wako Pure Chemical Industries, were used as organic solvents of the mobile phase in the high-performance liquid chromatography (HPLC) assays. Pure water (≤18.2 MΩ·cm), used as the mobile phase in the HPLC assay, was supplied by a Synthesis A10 water purification system followed by an Elix 10 water purification system (Millipore Corp., Billerica, MA, USA).
hot-water washing of the crude biodiesel to purify it. Recent studies have shown that the use of heterogeneous catalysts, such as metal oxides [2], ion-exchange resins [3,4] and immobilized lipases [5,6] can overcome these disadvantages. Solvent-free transesterification using a commercial immobilized lipase such as Novozym 435 (N435) can significantly reduce the amount of methanol required [5,6]. For biodiesel synthesis by N435, however, a single-phase reaction solution is needed at the beginning of the reaction, because phase-separated methanol inactivates N435. In order to add methanol up to the reaction equivalent, it must be added in two or three steps to prevent phase separation of the methanol. The most common form of biodiesel production using homogeneous alkali catalysts has two phases (methanol-rich and TGrich phases); and the addition of ethers such as tetrahydrofuran (THF) and liquefied dimethyl ether (DME) results in single-phase transesterification, thus significantly increasing the reaction rate [7,8]. If such cosolvents could be used in enzymatic biodiesel production, single-step addition of methanol up to the reaction equivalent would be possible, and the reaction would proceed much faster. In earlier works [9,10], a faster rate was observed in a transesterification system of TGs and methanol with N435 in the presence of those ethers. The effects of organic solvents except for alcohols (a substrate) on enzyme-catalyzed transesterification of vegetable oils have also been reported in earlier studies [10–13]. Unfortunately, the experimental conditions in those papers except for one paper [13], which were almost solvent-rich, differ significantly from those of actual biodiesel synthesis. The actual synthesis should use the amount of co-solvents to be minimized for a single-phase transesterification, because the co-solvents must be recovered with a distillation after the transesterification. Talukder et al. [13] reported the effect of mixing ratio of co-solvent to oil on reaction yield. However, the amount of co-solvents required to be added for the single-phase formation of a reaction system and the reaction yield in such a single-phase system are unknown. Moreover, although aggregation of N435 occurs during biodiesel synthesis due to the adsorption of glycerin to the surface of the enzyme-resins and is also affected by what the reaction vial is made of [14], the effect of co-solvent on the aggregation and glycerin phase has hitherto not been investigated. In this study, the transesterification of triolein and methanol in the presence of several selected co-solvents such as THF, acetone, and hexane, was performed using N435. The relationships between the reaction time and reaction yield during single-phase transesterification were examined. Simultaneously, dispersion of N435 and glycerin byproduct was observed, and the effects of co-solvents were investigated and contrasted with our observations of dispersion in previous studies on transesterification without co-solvents [14]. To better understand the effects of co-solvents on the dispersion and the reaction performance, in addition, phase equilibrium composition during the reaction progress was estimated using the LLE-UNIFAC model. We also attempted to calculate the potential for the transfer of water, glycerin, and methanol between the initial bulk solution and the water layer on the enzyme surface, and, using the calculated results, to investigate the reason for the inactivation caused by addition of hexane.
2.2. Experimental procedure In our previous study on an enzymatic transesterification of triolein and methanol [14], a conventional stepwise batch transesterification (two-step addition of methanol up to the reaction equivalent), as reported by Watanabe et al. [6], was employed. In the presence of cosolvents, the two-step addition of methanol was changed to one-step addition. The experimental procedure is, briefly, as follows. First, 0.4 g of N435 was placed in a 50-mL glass vial, followed by 9.65 g of triolein and an aliquot of co-solvent. In this study, tetrahydrofuran (THF), acetone, and hexane were selected as the co-solvents, because Talukder et al. [13] reported that THF and acetone were active co-solvents, whereas hexane was not. However, their experimental conditions as well as research objective differed from ours. We focus on minimizing the amount of co-solvents, which must be removed by distillation after the transesterification, and also their effect on N435 aggregation. Therefore, we examined the amount of each co-solvent required to keep the initial reaction system homogeneous. As a result, the amount of THF, acetone, and hexane added into the reaction system were 1.64, 3.33, and 5.6 g, respectively. Finally, methanol was slowly loaded, ensuring no direct contact with the N435. The molar ratio of methanol to triolein was three, namely the reaction equivalent, according to a report by Watanabe et al. [6]. The ratio is considered to be sufficient for the reaction, because we also observed a high yield of 93.2% in our previous work [14]. The weight fractions of all components at the beginning of the reaction are summarized in Table S1. The vial was immersed in a thermostatically-controlled water bath at 30 °C, and shaken at 130 rpm. At appropriate intervals, e.g., 2, 4, 11 h, etc., 100 µL of the reaction solution was sampled with a micro syringe. The sample was then diluted with isopropanol and the concentrations of triolein, diolein, monoolein, and methyl oleate in the sample were determined using a HPLC-UV system with a 3.0 × 150 mm ODS column as described in our previous study on the transesterification of triolein with a homogeneous alkali catalyst [8]. In addition, the concentrations were converted to the mole fraction (x) of methyl oleate (ME), monoolein (MO), diolein (DO), and triolein (TO). The yield of methyl oleate (YME) was calculated by the mole fraction and stoichiometric factor of each compound in the transesterification of TO, DO, and MO as follows.
YME =
2. Experimental section
xME 3xTO+2xDO+xMO+xME
(1)
2.1. Materials 3. Calculations by UNIFAC model Novozym 435 (N435) was purchased from Novozymes Japan Ltd. (Chiba, Japan). The enzyme, from Candida antarctica, was immobilized on particles of acrylic resin. The supplier reports its activity to be 10,000 U/g. Triolein (technical grade) and methanol (99.7%) used in the transesterification process were purchased from Kanto Chemical Co., Inc. (Tokyo, Japan) and Wako Pure Chemical Industries, Ltd. (Tokyo, Japan), respectively, and were used without further purification. A preliminary analytical test showed the oleic acid content of the triolein to be 0.564 wt%. Tetrahydrofuran (99.8%), acetone (99.5%),
The UNIFAC (UNIversal quasichemical Functional group Activity Coefficients) model is a group contribution method based on molecular thermodynamics [15], that is widely used to predict nonideality, namely the activity coefficient, in nonelectrolyte mixtures. The model describes the activity coefficient γi of component i by a combination of the combinatorial contribution due to differences in molecular size and shape, and the residual contribution due to differences in intermolecular interaction: 2
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Table 1 UNIFAC group assignment in this study. Compound
UNIFAC group assignment
Triolein Methyl oleate Glycerin Methanol THF Acetone Hexane
3 2 1 1 3 1 2
× × × × × × ×
CH3, 41 × CH2, 1 × CH, 3 × CH]CH, 3 × CH2COO CH3, 13 × CH2, 1 × CH]CH, CH2COO CH, 2 × CH2, 3 × OH CH3, 1 × OH CH2, 1 × FCH2O CH3, 1 × CH3CO CH3, 4 × CH2
lnγi = lnγiComb. + lnγiRes.
(2)
Many UNIFAC models have been reported since 1975. In our previous works [16,17], we reported the LLE-UNIFAC model [18] to be useful in terms of predicting the liquid-liquid equilibrium (LLE) in the biodiesel production process. Therefore, it was employed in the present study. In the UNIFAC model, the molecules in a system are divided into several groups, as shown in Table 1, after which the activity coefficient γi of component i is calculated according to the interaction between the groups and their size parameters. The size and interaction parameter values were cited from the LLE-UNIFAC parameter tables [18]. If the activity coefficient is calculated from the UNIFAC model, LLE can be obtained by solving the following mass balance and equilibrium equations derived from the criterion for phase equilibrium that the fugacity of component i in one phase is equal to that in the other phase: Fig. 1. Schematic illustration of inactivation of enzyme by methanol (MeOH) and glycerin (Gly), and three types of transfer free energy (ΔGtr) of MeOH, Gly, and water (W) between initial bulk solution (IBS) and the water layer on the enzyme surface (WL).
N
1=
∑ x iJ
(3)
i= 1
For the criterion for LLE,
γiI·x iI = γiII·x iII
ΔGi,tr B→A = μiA − μiB = μiA, * − μiB,*
(4)
B, ∞
γ = μio + RTlnγiA, ∞ − (μio + RTlnγiB,∞) = −RTln ⎜⎛ iA, ∞ ⎞⎟ γ ⎠ ⎝ i
where x refers to the mole fractions of component i. Superscripts I and II denote the liquid phase in a two-liquid system. For the LLE calculation, xiI and xiII were predicted by solving Eqs. (2), (3), and (4) and an additional constraint condition, which is an overall mass balance equation (the lever rule and feed composition) using the Newton-Raphson technique. As described in Section 4.2, we evaluated the equilibrium compositions of the oil phase and the glycerin phase that emerged during the transesterification reaction. To evaluate the variation of the phase compositions during the progress of the transesterification reaction, we first calculated the mole amounts of reactants and products such as TO, methanol, ME, and glycerin at a given methyl oleate yield (YME) and then, with the mole amount of co-solvent (acetone or THF), two phase LLE between glycerin phase and oil phase were calculated. Here, we assumed that N435 did not affect the phase equilibria and neglected the presence of esters other than ME. The mole fractions in the oil phase and glycerin phase, xiOil and xiGly, respectively were determined using Eqs (2)–(4), and the amount and equilibrium compositions of the two phases were estimated. Moreover, the transfer free energy of component i from B and A (ΔGtr i,B→A) was calculated as follows: A
The superscript * denotes a standard state (at infinite dilution). ΔGtr i,B→A can be calculated from the activity coefficients in solvents A and B estimated using the UNIFAC model. If a co-solvent leads to inactivation with several hours after the beginning of transesterification, the structure of the enzyme may be affected; e.g., as shown in Fig. 1a, inactivation is caused by increased methanol concentration and decreased water content on the surface of the enzyme, as reported by Fu and Vasudevan [10]. In addition, another inactivation due to adsorption of glycerin on the enzyme [20,21] may be promoted as shown in Fig. 1a. As shown in Fig. 1b–d, therefore, we evaluated the potential of this inactivation using the following three parameters: ΔGtr MeOH,IBS→WL: transfer free energy of methanol (MeOH) in the initial bulk solution (IBS) to the water layer on the enzyme surface (WL), ΔGtr Gly,IBS→WL: transfer free energy of glycerin (Gly) in the initial bulk solution (IBS) to the water layer on the enzyme surface (WL), and Δ Gtr W,WL→IBS: transfer free energy of water (W) from WL to IBS. Both parameters are given by: IBS
IBS
⎛ xMeOH ·γMeOH ⎞ tr ΔGMeOH,IBS → WL = − RTln ⎜ WL WL ⎟ ⎝ xMeOH ·γMeOH ⎠
(7)
B
tr A B o A o B ΔGi,B → A = μ i − μ i = μ i + RTlnx i γi − (μ i + RTlnx i γi )
tr ΔGGly, IBS → WL = − RTln
B
B ⎛ x i γi ⎞ = −RTln ⎜ x AγA ⎟ ⎝ i i ⎠
(6)
(5)
IBS IBS ⎛ x Gly ·γGly ⎞ ⎜ x WL ·γ WL ⎟ ⎝ Gly Gly ⎠
WL WL ⎛ x W ·γW ⎞ tr ΔGW, WL → IBS = − RTln ⎜ IBS IBS ⎟ ⎝ x W ·γW ⎠
where μi refers to the chemical potential of component i in each phase. The superscript o denotes a standard state (pure liquid of component i). If the conentration can be assumed to be at infinite dilution, it can be calculated from its infinite dilution activity coefficients (γi∞) in both phases [19]:
(8)
(9)
In the calculation of activity coefficient, we assumed that methanol or glycerin in WL and glycerin or water in IBS are in infinite dilution. Furthermore, WL is defined as pure water (xWWL = 1 and γWWL = 1). 3
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than those in the absence of co-solvent [14], namely under conventional solvent-free conditions. In contrast, YME values in the case of either THF or acetone were much higher than those under conventional conditions. Although there was no difference in the YME value at 2 h between hexane and acetone, inactivation was clearly observed after 4 h in the case of hexane. This indicates that not only methanol but also glycerin may be responsible for this inactivation. The reason for the inactivation is discussed in Section 4.3. With the addition of THF or acetone, YME values at 74 h exceeded 0.80, while YME at the same time under the conventional conditions reached only 0.623. In terms of reaction activity, THF and acetone were active co-solvents, while hexane was a negative co-solvent. This was the same as reported by Talukder et al. [13]. The active co-solvents significantly enhanced the reaction rate over 11 h. For example, the difference in reaction time to achieve the yield of 0.6 between with and without active co-solvent was over 2 days. We therefore examined the dispersion of N435 particles under each condition. Fig. 3a–c shows photographs of the particles during transesterification of triolein from 2 to 24 h. In the absence of co-solvent [14], no clear aggregation of the particles was observed up to 2 h. After 2 h, bulky and thin-layer aggregates were observed: the former aggregates grew with increasing reaction time. We speculated that the glycerin by-product acted as a form of adhesive in the interstices of the immobilized enzyme particles. In the presence of THF as well as acetone, no such bulky aggregates were observed. These results demonstrate that co-solvents play a role in preventing the aggregation of enzyme particles. In other words, the surface area of N435 was kept almost constant by the addition of co-solvent. This is likely the reason for the better reaction yield and rate in the presence of active co-solvents. The effect is very advantageous in terms of reaction rate and yield, because our previous work showed the bulky aggregates to have a very low reaction activity [14]. From 11 to 24 h, in the case of THF, however, small rigid or gel-like glycerin droplets coated with several particles, rather than aggregates of N435, were clearly observed, as shown in Fig. 3a. On the other hand, there were soft glycerin phases that were able to grow in the presence of acetone. From an industrial application perspective, acetone is better, since the glycerin phase was completely separated from the methyl oleate phase and N435 particles in the presence of acetone, as shown in Fig. 3b. This conclusion is different from that of an earlier work on the co-solvent effect of THF and acetone [13], because it focused on only reaction yield. Our previous study [14] showed the material of which the reaction vial was made strongly affected the aggregation of N435 and the reaction yield. Indeed, the use of a polypropylene vial produced only bulky aggregate (clumps of densely packed particles) and a reaction yield of around 0.45. In this study, therefore, the same transesterification in the presence of THF and acetone was performed using a 50-mL polypropylene vial. The results are shown in Fig. 4 and Table 3. The reaction curves were almost the same as those using a glass vial, showing that, in the presence of active co-solvents, the material of which the vial was made had little effect on the reaction. This can remove a negative factor from the reaction and is also an advantage from an industrial application perspective. Our observations on the effects of co-solvents on reaction yield significantly differed from those in earlier works [10–12], where immobilized lipases were used under solvent-rich conditions. Under solvent-rich conditions, hexane proved an effective solvent for enzymecatalyzed transesterification. In contrast, though, THF inactivated the activity of the enzyme, revealing that the solvent composition in the reaction system has a strong effect on the activity of lipase in N435. It should be noted that lipase activity under solvent-rich conditions cannot be used for predicting activity under solvent-poor conditions, such as those employed in the present study. The reason for the inactivation in the case of hexane, which is contrary to the earlier works, will be discussed in Section 4.3 using a thermodynamic approach.
Fig. 2. Reaction yield (YME) versus reaction time (t) in the enzyme-catalyzed transesterification of triolein in the presence of co-solvents (hexane, tetrahydrofuran (THF), and acetone) using a 50-mL glass vial at 30 °C. *: our previous work [14], (gv): glass vial.
From these assumptions, Eqs. (7)–(9) are transformed: IBS
IBS
⎛ xMeOH ·γMeOH ⎞ tr ΔGMeOH,IBS → WL = − RTln ⎜ ⎟ WL, ∞ γMeOH ⎝ ⎠
(10)
IBS,∞
tr ΔGGly, IBS → WL = − RTln
⎛ γGly ⎞ ⎜ γ WL, ∞ ⎟ ⎝ Gly ⎠
(11)
⎛ 1 ⎞ tr ΔGW,WL → IBS = − RTln ⎜ IBS, ∞ ⎟ ⎝ γW ⎠
(12)
where superscript ∞ refers to infinite dilution. The effect of co-solvents on WL is discussed based on these parameters. 4. Results and discussion 4.1. Effect of co-solvents on the reaction yield and immobilized enzyme particles The reaction yields of methyl oleate (YME) with or without co-solvents are plotted as a function of the reaction time (t) as shown in Fig. 2 and Table 2. YME values in the presence of hexane were much lower Table 2 Reaction yield (YME) with and without co-solvents (hexane, tetrahydrofuran (THF), acetone) during methanolysis of triolein by Novozym 435 in a glass vial. t/h
YME with hexane/–
YME with THF/–
YME with acetone/–
YME without cosolvent*/–
2 4 11 24 48 64 72 74 94 96
0.044 0.067 n.m. 0.081 n.m. n.m. n.m. n.m. n.m. n.m.
n.m. 0.266 0.381 0.681 0.778 n.m. 0.826 n.m. n.m. n.m.
0.049 0.109 0.375 0.603 0.768 0.835 0.832 n.m. n.m. 0.846
0.136 0.197 0.344 0.438 0.516 n.m. n.m. 0.628 0.685 nm
*Our previous work according to a conventional solvent-free system [14], n.m.: not measured. 4
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Fig. 3. Novozym 435 particles during the transesterification of triolein with and without co-solvent using a 50-mL glass vial. *: our previous work [14].
Table 3 Reaction yield (YME) with and without co-solvents (hexane, tetrahydrofuran (THF), acetone) during methanolysis of triolein by Novozym 435 in a polypropylene vial. t/h
YME with THF/–
YME with acetone/–
YME without co-solvent*/–
2 4 11 24 46 64 72 74 94 96
n.m. 0.255 0.459 0.725 0.872 n.m. 0.893 n.m. 0.897 n.m.
0.042 0.106 0.354 0.608 0.760 0.815 0.831 n.m. n.m. 0.843
0.142 0.190 0.306 0.373 0.421 n.m. n.m. 0.447 0.454 n.m.
*Our previous work according to a conventional solvent-free system [14], n.m.: not measured.
estimated using the LLE-UNIFAC model [18]. Here, we assumed that there were only two phases, oil phase and glycerin phase, and neglected the effect of N435 on the LLE. The detailed calculation method was already described in the Section 3. Methanol and co-solvents were assumed to dissolve in either or both of the oil phase and the glycerin phase, and there was no third phase. The temperature was set to 30 °C and the initial amounts of triolein, methanol, and co-solvents were the same as those provided in Section 2. For four fixed methyl oleate yields (YME) from 0.3 to 0.9, the equilibrium compositions were determined. The calculated results for the oil and glycerin phases are shown in Fig. 5a, b. For the oil phase, the concentrations of reactants such as methanol and triolein in the case of THF exceeded those in the case of acetone. The addition of acetone led to more diluted concentrations of both reactants. The reaction rate in the presence of THF therefore
Fig. 4. Reaction yield (YME) versus reaction time (t) in the enzyme-catalyzed transesterification of triolein in the presence of co-solvents (hexane, tetrahydrofuran (THF), and acetone) using a 50-mL polypropylene vial at 30 °C. *: our previous work [14], (pv): polypropylene vial.
4.2. Estimation of the phase equilibrium of the reaction system Although both THF and acetone were effective co-solvents for the transesterification of triolein, THF was superior in terms of enhancing the reaction rate, as shown in Figs. 2 and 4. Meanwhile, acetone was superior to THF with respect to separation of the glycerin by-product, as shown in Fig. 3a, b. To better understand these differences, variation in phase equilibrium composition during the transesterification with co-solvent was 5
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Fig. 5. Estimated compositions of oil phase in equilibrium with glycerin phase with THF or acetone as a cosolvent, during the transesterification of triolein calculated using the LLE-UNIFAC model [18]. a) Mole fraction of component i in the oil phase (xiOil) and glycerin phase (xiGly) at reaction yields (YME) of 0.3, 0.5, 0.7 and 0.9, in the presence of THF as a co-solvent. b) Mole fraction of component i in the oil phase (xiOil) and glycerin phase (xiGly), at reaction yields (YME) of 0.3, 0.5, 0.7 and 0.9, in the presence of acetone as a co-solvent.
to cause the methanol-driven inactivation and/or the glycerin-driven inactivation. Table 4 shows the transfer free energies of methanol and glycerin from the initial bulk solution to the water layer on the enzyme tr surface (ΔGtr MeOH,IBS→WL and ΔGGly,IBS→WL, respectively) and the activity coefficients used for calculating the energy for all the reaction systems. In the calculated results of ΔGtr MeOH,IBS→WL, THF and hexane have lower values. This suggests that both solvents have higher potentials of the methanol-driven inactivation than acetone and non-co-solvent condition. Furthermore, the results of ΔGtr Gly,IBS→WL indicate that it is easier to bring about the glycerin-driven inactivation in the presence of hexane. Therefore, we considered that the addition of hexane has a larger negative impact on the enzymatic transesterification in terms of methanol- and glycerin-driven inactivations than the other co-solvents. Although the ΔGtr Gly,IBS→WL value for hexane is equal to that for the nonco-solvent condition, the reaction rate without hexane is expected to be much faster than that with hexane because the calculated activities of TO and MeOH in the absence of hexane are much higher as shown in Table 4. The larger amounts of products such as ME and glycerin due to the high reaction rate might mitigate the two inactivations. Indeed, the conventional two-step addition of methanol [6] demonstrates that an increase of ME concentration is able to dissolve more methanol in bulk solution. This approach using the UNIFAC model may thus be useful for evaluating co-solvent effects. We also estimated the possibility that cosolvents destabilize the water layer. The transfer free energy of water
appears to be faster than that with acetone as shown in Fig. 4. The content of co-solvents in the glycerin phase differed significantly, with the concentration of acetone being much higher than that of THF. Since acetone is a less viscous liquid, the high concentration of acetone resulted in a soft glycerin phase. Reducing the viscosity of the glycerin phase was useful for removing viscous glycerin from the reaction system. In contrast, the concentration of THF in the glycerin phase was much lower, indicating that the glycerin phase was more viscous. This is a fairly good explanation for the formation of the small rigid glycerin phases coated with N435 particles shown in Fig. 3b, a. If the removal of the glycerin phase is the key factor in biodiesel production with N435, acetone is superior to THF as a co-solvent. 4.3. Estimation of transfer free energy of methanol and water It is well known that contact with phase-separated methanol inactivates the immobilized lipase. It is thought that hydrophilic methanol molecules remove the water molecules on the surface of the lipase [10]. Moreover, adsorption of glycerin on the lipase serves as a barrier to transport reactants resulting in a kind of inactivation of the lipase [20,21]. Since the inactivation clearly occurred after 4 h in our experiments using hexane, the effect of the produced glycerin should also be considered in addition to the methanol-driven inactivation. Therefore, we tried to investigate whether the addition of hexane tends 6
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potentially useful for evaluating the effects of co-solvents on enzymatic transesterification.
Table 4 Activity coefficients of methanol (MeOH), glycerin (Gly) and water (W) in the initial bulk solution (IBS) and the water layer on the enzyme surface (WL) estimated by LLE-UNIFAC model [18], mole fraction of MeOH in ISB, activity of MeOH and TO in ISB, and transfer free energy of MeOH, Gly, and W between IBS and WL calculated by Eqs. (10)–(12).
γMeOHIBS/– γGLyIBS, ∞/– γMeOHWL, ∞/– γGLyWL, ∞/– γWIBS, ∞/– xMeOHIBS/– aMeOHIBS/– aTOIBS/– −1 ΔGtr MeOH,IBS→WL/kJ mol −1 ΔGtr Gly,IBS→WL/J mol −1 ΔGtr W,WL→IBS/kJ mol
With THF
With acetone
With hexane
No cosolvent
1.98 91.2 2.24 0.583 10.7 0.492 0.973 0.0743 2.10 −12.7 5.97
1.92 49.0 2.24 0.583 9.73 0.324 0.622 0.116 3.22 −11.2 5.73
3.04 271 2.24 0.583 29.2 0.302 0.917 0.0137 2.25 −15.5 8.50
1.60 275 2.24 0.583 17.2 0.500 0.801 0.467 2.58 −15.5 7.16
Acknowledgement This work was supported by JSPS (the Japan Society for the Promotion of Science) KAKENHI Grant Number 21360449. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.fuel.2019.116600. References [1] R REN21, Renewables 2018 GLOBAL STATUS REPORT, http://www.ren21.net/wpcontent/uploads/2018/06/178652_GSR2018_FullReport_web_-1.pdf [accessed October 1, 2018]. [2] Kouzu M, Hidaka J. Transesterification of vegetable oil into biodiesel catalyzed by CaO: a review. Fuel 2012;93:1–12. [3] Shibasaki-Kitakawa N, Honda H, Kuribayashi H, Toda T, Fukumura T, Yonemoto T. Biodiesel production using anion ion-exchange resin as heterogeneous catalyst. Bioresour Technol 2007;98:416–21. [4] Shibasaki-Kitakawa N, Tsuji T, Chida K, Kubo M, Yonemoto T. Simple continuous production process of biodiesel fuel from oil with high content of free fatty acid using ion-exchange resin catalysts. Energy Fuels 2010;24:3634–8. [5] Shimada Y, Watanabe Y, Samukawa T, Sugihara A, Noda H, Fukuda H, et al. Conversion of vegetable oil to biodiesel using immobilized Candida antarctica lipase. JAOCS 1999;76(7):789–93. [6] Watanabe Y, Shimada Y, Sugihara A, Noda H, Fukuda H, Tominaga Y. Continuous production of biodiesel fuel from vegetable oil using immobilized Candida antarctica lipase. JAOCS 2000;77(4):355–60. [7] Boocock DGB, Konar SK, Mao V, Sidi H. Fast one-phase oil-rich processes for the preparation of vegetable oil methyl esters. Biomass Bioenergy 1996;11:43–50. [8] Kuramochi H, Maeda K, Osako M, Nakamura K, Sakai S. Superfast transesterification of triolein using dimethyl ether and a method for high-yield transesterification. Ind Eng Chem Res 2008;47:10076–9. [9] Maeda K, Kuramochi H, Arafune K, Itoh K, Yamamoto T. Transesterification of triolein and methanol by Novozym 435 with dimethyl ether. J Chem Eng Jpn 2017;50(12):924–8. [10] Fu B, Vasudevan PT. Effect of organic solvents on enzyme-catalyzed synthesis of biodiesel. Energy Fuels 2009;23:4105–11. [11] Gog A, Roman M, Toşa M, Paizs C, Irimie FD. Biodiesel production using enzymatic transesterification – Current state and perspectives. Renewable Energy 2012;39:10–6. [12] Guldhe A, Singh B, Mutanda, Permaul K, Bux F. Advances in synthesis of biodiesel via enzyme catalysis: novel and sustainable approaches. Renew Sustain Energy Rev 2015;41:1447–64. [13] Talukder MMR, Puah SM, Wu JC, Won CJ, Chow Y. Lipase-catalyzed methanolysis of palm oil in presence and absence of organic solvent for production of biodiesel. Biocatal Biotransform 2006;24(3):257–62. [14] Kuramochi H, Kobayashi T, Maeda K. Aggregation of immobilized enzyme during transesterification of triolein and methanol, and the effect of two types of aggregates on reaction yield. Fuel 2020;260:116343. [15] Fredenslund A, Jones RL, Prausnitz JM. Group-contribution estimation of activity coefficients in non-ideal liquid mixtures. AIChE J 1975;21:1086–99. [16] Kuramochi H, Maeda K, Kato S, Osako M, Nakamura K, Sakai S. Application of UNIFAC models for prediction of vapor-liquid and liquid-liquid equilibria relevant to separation and purification processes of crude biodiesel. Fuel 2009;8:1472–7. [17] Maeda K, Kuramochi H, Asakuma Y, Fukui K, Tsuji T, Osako M, et al. De-emulsification of biodiesel mixtures with dimethyl ether. Chem Eng J 2011;169:226–30. [18] Magnussen T, Rasmussen P, Fredenslund A. UNIFAC parameter table for prediction of liquid-liquid equilibria. Ind Eng Chem Process Des Dev 1981;20:331–9. [19] Kobayashi T, Kuramochi H, Xu K-Q, Maeda K. Simple solvatochromic spectroscopic quantification of long-chain fatty acids for biological toxicity assay in biogas plants. Environ Sci Pollut Res [in press]. [20] Dossat V, Combes D, Marty A. Continuous enzymatic transesterification of high oleic sunflower oil in a packed bed reactor: influence of the glycerol production. Enzyme Microb Technol 1999;25:194–200. [21] Xu Y, Nordblad M, Nielsen MP, Brask J, Woodley JM. In situ visualization and effect of glycerol in lipase-catalyzed ethanolysis of rapeseed oil. J Mol Catal B Enzym 2011;72:213–9.
γMeOHIBS: activity coefficient of MeOH in IBS, γGlyIBS, ∞: infinite dilution activity coefficient of Gly in IBS, γMeOHWL, ∞: infinite dilution activity coefficient of MeOH in WL, γGlyWL, ∞: infinite dilution activity coefficient of Gly in WL, γWIBS, ∞ : infinite dilution activity coefficient of W in IBS, xMeOHIBS: mole fraction of MeOH in ISB, aMeOHIBS: activity of MeOH in ISB, aTOIBS: activity of TO in tr ISB,ΔGtr MeOH,IBS→WL: transfer free energy of MeOH from IBS to WL, GGly,IBS→WL: transfer free energy of Gly from IBS to WL,ΔGtr W,WL→IBS: transfer free energy of W from WL to IBS.
from the water layer to the bulk solution (ΔGtr WL,W→IBS) was calculated. The results are shown in Table 4. All the values are high positive values, meaning that water molecules on the enzyme surface are very unlikely to transfer to the initial bulk solution. The present co-solvents are considered to have little effect on the water layer if the transfer of methanol is ignored. 5. Conclusions We investigated the effects of three co-solvents on the enzyme-catalyzed transesterification of triolein and methanol. Hexane led to inactivation of Novozym 435. However, the reaction yield in the presence of acetone or tetrahydrofuran (THF) was found to be much higher than without co-solvent. The results were almost the same as reported in an earlier work. However, we first revealed the other effects due to the cosolvents as follows: both active co-solvents inhibited the aggregation of Novozym 435 particles that occurred in the absence of the co-solvents, resulting in enhanced reaction rate and yield. In addition, there was a clear difference in the formation and dispersion of the glycerin phase between THF and acetone: viscous and rigid glycerin droplets coated with Novozym 435 particles in the case of THF, and soft glycerin phases, which can form a single phase, in the case of acetone. Therefore, acetone is useful for removing the glycerin phase from the reaction system. The phase equilibrium composition in the reaction system with co-solvents, estimated using the LLE-UNIFAC model, indicated that acetone significantly dilutes the glycerin phase. Finally, the transfer free energy of methanol, glycerin, and water between the initial bulk solution and water layer on the enzyme surface was estimated using the LLE-UNIFAC model. The lower transfer energies of methanol and glycerin from the bulk to the water layer in the presence of hexane indicate that hexane has a higher potential of the methanol-driven and glycerin-driven inactivations. This may be the reason why hexane led to a lower reaction yield. Analysis based on the LLE-UNIFAC model is thus
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