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Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L
Renewable Energy xxx (xxxx) xxx
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Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L Shangde Sun*, Kaiyue Li Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road 100, Zhengzhou, 450001, Henan Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2019 Received in revised form 29 October 2019 Accepted 2 November 2019 Available online xxx
Biodiesel is a green and sustainable energy, which is a fatty acid alkyl ester as an alternative to petroleum diesel. In the work, phoenix tree seed oil, one kind of undeveloped woody plant resource, was used as the novel raw material for biodiesel preparation. Several free liquid lipases were used as biocatalysts to catalyze the transesterification of phoenix tree seed oil (PTSO) with methanol. Effects of transesterification variables (enzyme load, substrate ratio, transesterification time and temperature) on biodiesel preparation were evaluated and optimized using RSM. Results showed that PTSO was a good alternative for biodiesel preparation. Among these tested free liquid lipases, lipozyme TL100L from Thermomyces laguginosus showed the best performance for the transesterification. Transesterification variables were optimized and the maximum biodiesel yield (98.8 ± 1.1%) was achieved under the optimal conditions (enzyme load 10%, transesterification temperature 30 C, substrate ratio (PTSO to methanol) 1:5 (mol/mol) and reaction time 6.98 h). The activation energy of biodiesel formation was 21.3 kJ/mol. Kinetic parameters K’m and Vmax were 2.55 101 mol/L and 6.9 103 mol/(L$min), respectively. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel Lipase Phoenix tree seed oil Transesterification Response surface methodology Kinetic
1. Introduction Biodiesel, as a green and sustainable energy, is consisted of mono-alkyl esters, mainly the methyl esters of fatty acids, which is non-toxic, biodegradable, renewable, and environmental [1e3]. The materials used for biodiesel preparation in the previous reports were mainly edible oils (for example, soybean oil, flaxseed oil and cottonseed oil) [4e8]. Compared with these edible oils, some nonedible vegetable oils and waste cooking oils have shown some advantages as the alternative resource [9e11]. Recently, some microbial oils and industry wastes have also been developed as the raw materials for biodiesel preparation [12e14]. Phoenix tree seed is one kind of undeveloped woody plant resource, which can be obtained from Phoenix tree grown by the road and in the courtyard in China [15,16]. In our previous report, we found the oil content of the seeds was 27.8 ± 0.3% [15]. In phoenix tree seed oil (PTSO), oleic acid (O, 22.2%), palmic acid (P, 17.43%) and linoleic acid (L, 30.2%) were the main fatty acids. However, no information using PTSO as raw material to prepare
* Corresponding author. E-mail addresses: [email protected] (S. Sun), [email protected] (K. Li).
biodiesel was available. In the previous reports about biodiesel preparation, different catalysts were used to catalyze the transesterification of oils with methanol, for example, acids [17,18], alkalis (sodium hydroxide, potassium hydroxide) [19,20] and enzymes (for example, immobilized lipases and free lipases from Pseudomonas aeruginosa, Yarrowia lipolytica and Rhizomucor miehei) [21e25]. Compared with acid and alkali, due to the mild reaction conditions, enzyme has shown good performance for the transesterification of oils [26,27]. And in these previous enzymatic transesterification of oils, immobilized enzymes were often used to prepare biodiesel [28,29]. Recently, free liquid lipases have attracted more attention as the alternative biocatalyst for biodiesel preparation [30e33]. However, the relative information using free lipase lipozyme TL100L as biocatalyst is enormously uncommon. In the work, PTSO, as the novel raw material, was used for biodiesel preparation. Several free liquid lipases were used as biocatalysts to catalyze the transesterification. Effects of transesterification variables (substrate ratio, enzyme load, time and temperature) on biodiesel preparation were evaluated. The interaction of transesterification variables were also investigated and optimized by response surface methodology (RSM). Reaction thermodynamic and kinetics were also analyzed.
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Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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200 C at 2 C/min, and increased to 290 C at 20 C/min, and then increased to 320 C at 6 C/min and hold at 320 C for 1min, finally increased to 360 C at 20 C/min and hold at 360 C for 7min. Biodiesel yield was calculated as follows:
2. Materials and methods 2.1. Materials PTSO with 4.2 ± 0.5% free fatty acid content was prepared according to Soxhlet extraction method from phoenix tree seeds (Jiangsu, China). Free liquid lipases (lipase A from Candida sp. (CALA), Lipozyme from Thermomyces laguginosus (Lipozyme TL100L) and lipase B from Candida sp. (CALB) were provided by Novozymes A/S (Begsvaerd, Denmark).
Biodiesel yield ð%Þ ¼
mFAME 100% mTG þ mDG þ mMG þ mFAME þ mFA (1)
where m is the mass weight of product or reactants.
2.2. Enzymatic reaction of PTSO with methanol
2.4. RSM design
PTSO (2.50 g) was mixed with methanol (0.47 g) at 300 rpm and 30 C in 25-mL round-bottom flasks. Magnetic stirring bioreactor was used to mix reaction mixture and control the temperature for the reaction. The stirring speed was 300 rpm. When 0.20 g lipase was added into the mixture, the enzymatic transesterification was initiated. These standard conditions were used except when otherwise stated in the text. Samples (20 mL) were withdrawn using one 20 mL sample needle and dissolved in 3 mL n-hexane, and then dried using anhydrous sodium sulfate, and followed by centrifugation. Finally, the supernatant liquid was collected using one 2.5mL injector and filtered using one microfilter (0.45 mm) for GC analysis.
RSM with four factors and three levels was designed using BoxeBehnken in this work. The reaction variables and levels were transesterification temperature (20 C, 30 C, and 40 C), time (0.05, 7.00 and 13.95 h), enzyme load (1%, 8%, 15%; w/w) and substrate ratio (PTSO/methanol, 1:1, 5:1, 9:1 (mol/mol)) (Table 1).
2.3. Determination of biodiesel by GC
Y ¼ b0 þ
2.5. Statistical analysis From the RSM, 29 experiments were designed to evaluate the effect of four transesterification parameters on biodiesel yield. The quadratic polynomial equation between transesterification parameters with biodiesel yield was as follows: 4 X
bii X 2i þ
i1
Biodiesel samples were analyzed using GC (7890B) with a DB1ht capillary column (28 m 0.25 mm, 0.1 mm film thickness) and flame-ionization detector (FID). The flow of N2 was 4 mL/min. The temperatures of FID and injector were both 350 C. The column temperatures were firstly set at 170 C, secondly increased to
3 4 X X
bij Xi Xj
(2)
i1 jiþ1
where Y is biodiesel yield, Xi and Xj are the transesterification parameters, b0, bi, bii, and bij are the constant, linear, quadratic and cross term coefficients, respectively. To ensure the validity of the results, all experiments were
Table 1 RSM design and result of Lipozyme TL100L-catalyzed the transesterification of PTSO with methanol for biodiesel preparation. Treatmenta
Reaction time X1 (h)
Temperature X2 (oC)
Substrate ratio X3 (mol/mol)
Enzyme load X4 (%)
Biodiesel yield Y (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
7.00 (0) 7.00 (0) 0.05 (1) 13.95 (1) 7.00 (0) 0.05 (1) 7.00 (0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 0.05(-1) 7.00(0) 13.95(1) 0.05(-1) 7.00(0) 7.00(0) 7.00(0) 13.95(1) 13.95(1) 0.05(-1) 7.00(0) 0.05(-1) 7.00(0) 13.95(1) 13.95(1)
30 (0) 30 (0) 20 (1) 30 (0) 40 (1) 30 (0) 20 (1) 20(-1) 40(1) 20(-1) 30(0) 30(0) 30(0) 20(-1) 30(0) 30(0) 40(1) 40(1) 40(1) 30(0) 40(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 20(-1) 30(0)
5 (0) 1 (1) 5 (0) 9 (1) 5 (0) 5 (0) 1 (1) 9(1) 9(1) 5(0) 5(0) 1(-1) 5(1) 5(1) 1(-1) 9(1) 5(0) 5(0) 5(0) 9(1) 1(-1) 5(0) 5(0) 9(1) 5(0) 5(0) 5(0) 5(0) 1(-1)
8 (0) 1 (1) 8 (0) 8 (0) 1 (1) 15 (1) 8(0) 8(0) 8(0) 1(-1) 8(0) 15(1) 8(0) 15(1) 8(0) 1(-1) 8(0) 8(0) 15(1) 15(1) 8(0) 1(-1) 15(-1) 8(0) 8(0) 1(-1) 8(0) 8(0) 8(0)
95.7 ± 1.5 56.2 ± 1.0 76.3 ± 1.3 83.6 ± 1.2 18.6 ± 0.8 75.3 ± 1.4 68.6 ± 0.9 90.7 ± 1.1 55.9 ± 0.6 40.8 ± 1.7 95.9 ± 0.8 67.51 ± 1.0 94.0 ± 1.2 96.4 ± 0.8 34.8 ± 1.0 12.7 ± 0.9 96.7 ± 1.6 73.6 ± 1.3 95.7 ± 1.5 97.0 ± 3.0 67.4 ± 1.7 15.2 ± 0.7 96.0 ± 0.4 62.3 ± 1.0 94.0 ± 1.5 16.0 ± 0.9 97.1 ± 0.7 96.2 ± 1.7 48.2 ± 0.6
a
Numbers were run randomly.
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performed at least in triplicate. The results were expressed as averages ± S.E.M. The significance of the difference was estimated using a two-way analysis of variance (ANOVA). Statistical significance was considered at p < 0.05. 3. Results and discussion 3.1. Liquid lipases screening The effect of free liquid lipases (Lipozyme TL100L, CALA and CALB) on the transesterification was shown in Fig. 1a. Among these lipases, the maximum biodiesel yield (80.0 ± 2.2%) was achieved using Lipozyme TL100L, which is about 6 times that of CALA (12.2 ± 1.5%) and CALB (14.7 ± 1.9%). These results were ascribed to the good resistance to methanol and phospholipid of Lipozyme TL100L. Compared with Lipozyme TL100L, the intra-protein
3
hydrogen bonding interactions of CALA and CALB associated with catalytic activity can be easily weakened in methanol, which resulted in the deactivation of CALA and CALB. Similar result that the hydrogen bond (H-bond) related with catalytic activity of CALB destroyed by methanol was also found [34]. In the previous report, lipase from P. cepacia also showed good methanol resistance at high methanol concentration [35]. Fig. 1b also shows that the initial reaction rates of the transesterification catalyzed by CALA and CALB (2.2e2.5 103 mol/(L$min)) were almost half of Lipozyme TL100L (4.5 103 mol/(L$min)). The time to achieve equilibrium using Lipozyme TL100L as catalysts was 7 h, which was very shorter than some immobilized lipases, for examples, immobilized Candida sp. 99e125 (24 h at 40 C) [36,] and immobilized Candida antarctica lipase (24 h at 50 C) [37]. These were ascribed to the lower mass transfer limitation of free liquid Lipozyme TL100L than the immobilized lipases. These can also be confirmed by the effect of stirring. When the stirring speed was greater than 300 rpm, no obvious change of biodiesel yield was found. These also indicated that external mass transfer limitation on the reaction can be eliminated. 3.2. Effect of temperature When the temperature increased from 10 C to 30 C, initial reaction rate increased from 4.0 103 mol/(L$min) to 8 103 mol/(L$min) and biodiesel yield also increased from 73.7 ± 1.7% to 95.3 ± 1.9% at 7 h (Fig. 2). The time to reach equilibrium also shortened from 7 h to 3 h. These results were due to the decrease of mass transfer limitation, and the increase of catalytic activity of enzyme and the number of activated molecules at high temperature. However, when the temperature varied from 30 C to 60 C, a remarkable decrease of biodiesel yield from 95.3 ± 1.9% to 17.3 ± 1.3% was found. The initial reaction rate also decreased from 8 103 mol/(L$min) to 1.3 103 mol/(L$min) and the time to reach equilibrium increased from 3 h to 12 h. The result was attributed to the deactivation of Lipozyme TL 100 L at high temperature. Similar deactivation at high temperature can also be found in other enzymatic reactions [22]. A linear relationship between initial reaction rate (lnVo) with temperature (1/T) (Y ¼ 2.56443Xþ3.58363,R2 ¼ 0.93118) was found (Fig. 2c), and Arrhenius equation of biodiesel formation can be obtained from the linear equation as follows.
LnVo ¼
2564:43 3:58363 ½A
(3)
Accordingly, the activation energy (Ea) of biodiesel formation by the transterification of PTSO using free liquid lipase Lipozyme TL 100 L as catalyst was calculated as 21.3 kJ/mol. Compared with the report of Guat et al. [38], the activation energy of Lipozyme TL IM is 22.2 kJ/mol, which is higher than free lipase Lipozyme TL 100 L.The lower activation energy means shorter equilibrium time. Fig. 2 also shows that, the optimal reaction temperature of free liquid Lipozyme TL 100 L was 30 C, which was lower than the immobilized Lipozyme TLIM (lipase from Thermomyces laguginosus) (40 C) [39]. Compared with the free liquid Lipozyme TL 100 L, high optimal reaction temperature was due to the protection of the immobilization of lipase from Thermomyces laguginosus. Compared with other lipases (for example, lipozyme TL IM [39e41] and Candida rogues lipase [42,43]), the optimal reaction temperature of free liquid Lipozyme TL 100 L (30 C) was low, which showed that Liposome TL 100 L was the best choice for biodiesel preparation. Fig. 1. (a) Effect of different liquid lipases (Lipozyme TL100L, CALA and CALB) on biodiesel yield; (b) Effect of different liquid lipases (Lipozyme TL100L, CALA and CALB) on initial reaction rate. The reactions were conducted with 1:5 (mol/mol) substrate ratio, 8% (v/v) lipase load with different enzymes at 40 C.
3.3. Effect of substrate ratio Methanol has significantly effect on enzyme activity. Biodiesel
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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Fig. 2. (a) Effect of different reaction temperatures on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of different reaction temperatures on the initial reaction rates; (c) The relationship of initial reaction rate (lnV0) and reaction temperature (1/T). The reactions were conducted with 8% (v/v) Lipozyme TL 100 L lipase load and 1:5 (mol/mol) substrate ratio.
yield increased when substrate ratio (PTSO/methanol) changed from 1:4 to 1:5 (Fig. 3a). However, with further increase in methanol ratio from 1:5 to 1:15, biodiesel yield significantly decreased from 95.3 ± 1.9% to 55.7 ± 2.4%. Meanwhile, the initial reaction rate (Fig. 3b) also decreased from 6.9 103 mol/(L$min) to 2.2 103 mol/(L$min) and the time to reach equilibrium increased from 3 h to 7 h. The shortest time to reach equilibrium was 3 h. These were attributed to the deactivation of enzyme with high methanol concentrations [44]. Similar effect of methanol can also be found in other enzymatic reactions [45,46]. For example, the structure of Yarrowia lipolytica lipase was disrupted when methanol concentrations were higher than 30% [23]. The catalyst activity of Callera™ Trans L lipase from Thermomyces lanuginosus decreased when the molar ratio of triglyceride (degummed soybean oil) to methanol was 1:4.5 [47]. The catalyst activity of immobilized Candida rugosa lipase decreased when the substrate ratio of oil to methanol was lower than 1:4 [43]. Compared with these lipases, Lipozyme TL100L has high catalyst activity at 1:5 molar ratio of oil to methanol (Fig. 3). The methanol concentration was higher than those in the previous reports, which indicated that Lipozyme TL
100 L has high methanol tolerance in this reaction system. 3.4. Effect of enzyme load Fig. 4 shows that, biodiesel yield increased from 17.8 ± 0.7% to 95.2 ± 1.3% with enzyme load ranging from 1% to 10%, and the initial reaction rate also increased from 1.4 103 mol/(L$min) to 9 103 mol/(L$min) and the time to reach equilibrium also shortened from 7 h to 3 h. The maximum biodiesel yield (95.2 ± 1.3%) was obtained with 10% Lipozyme TL100L load at 7 h, which were ascribed to the presence of more effective catalytic sites at high enzyme load. However, when enzyme load increased from 10% to 15%, the initial reaction rate decreased from 9 103 mol/(L$min) to 12 103 mol/(L$min), however, the biodiesel yield also decreased from 95.2 ± 1.3% to 89.1 ± 2.1%, which could be due to the aggregation of Lipozyme TL100L and diffusion limitation of reaction system at high enzyme load. Similar results that, the catalyst efficiency per unit of enzyme decreased when excess enzyme was present in reaction system, can also be found in other enzymatic reactions [48,49]. After reaction was completed,
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Fig. 3. (a) Effect of substrate ratio on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of substrate ratio on initial reaction rates. The reactions were conducted with 8% (v/v) Lipozyme TL 100 L load at 25 C.
the free lipase Lipozyme TL 100 L was separated and reused. However, biodiesel yield decreased by 45.5 ± 1.8% at the 2nd use of the lipase.
Fig. 4. (a) Effect of enzyme load on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of enzyme load on initial reaction rate. The reactions were conducted with 1:5 (mol/mol) substrates ratio at 30 C.
3.5. Model fitting To further improve the reaction efficiency for biodiesel preparation, the interaction of the transesterification variables were evaluated using RSM. For biodiesel preparation, the high coefficient (R2) of the model (0.9126) (Table 2) showed that the transesterification model was significant. Therefore, biodiesel yield can be satisfactorily explained by the following quadratic polynomial model: Biodieselyield (%) ¼ 3.97X1 þ 1.22X2 þ 13.57X3 þ 6.53X4 þ 0.01X1X2 þ 0.07X1X3 þ 0.11X1X4 - 0.21X2X3 þ 0.08X2X4 þ 0.65X3X4 - 0.31X21 - 0.02X22 - 1.17X23 - 0.53X24 -14.08 (4) The equation showed that X1, X2, X3, X4,X1X2, X1X3,X1X4,X2X4and X3X4 had positive effects on biodiesel yield. However, other parameters have negative effects on the transesterification.
Fig. 5 shows that the effect of reaction parameters on biodiesel yield decreased as substrates ratio > enzyme load > time > temperature. The effect of interaction of temperature, time, substrate ratio and enzyme load on biodiesel yield can be better evaluated using the 3D graphs. As shown in Fig. 5a, the optimal biodiesel yield appeared at 20 Ce30 C and 7 he10.48 h, and the effect of transesterification time on biodiesel yield was greater than reaction temperature. Similarly, the effects of enzyme load and substrate ratio on biodiesel yield were greater than that of reaction temperature (Fig. 5b and c). The optimal biodiesel yield appeared at 7.00 he10.48 h and substrate ratio from 1:3 to 1:7 with 11%e13% enzyme load (Fig. 5d and e). According to the RSM results, the effects of substrate ratio and enzyme load on biodiesel yield were higher than reaction time. The interaction (P ¼ 0.0087 < 0.05) of substrates ratio and enzyme load had a significant effect on biodiesel yield (Fig. 5f). The
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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Table 2 ANOVA analysis for response surface model of biodiesel yield. Source
Sum of squares
Degree of freedom
Mean square
F value
Prob > F
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 Residual Lack of fit Pure error Total
20938.1 793.8 311.1 294.9 11310.5 2.6 15.6 115.6 282.2 115.6 1331.9 2005.6 1998.4 7.1 22943.7
14 1 1 1 1 1 1 1 1 1 1 14 10 4 28
1495.58 793.8 311.1 294.9 11310.5 2.6 15.6 115.6 282.2 115.6 1331.9 143.3 199.8 1.78 R2 ¼ 0.9126
10.44 5.54 2.17 2.06 78.95 0.018 0.11 0.81 1.97 0.81 9.30
<0.0001 0.0337 0.1627 0.1733 <0.0001 0.8956 0.7463 0.3843 0.1822 0.3843 0.0087
112.08
0.0002
optimal biodiesel yield appeared at 9%e15% enzyme load and 1:5 to 1:9 substrates ratio. When substrates ratio and enzyme load were lower than the optimum values (substrates ratio 1:5, enzyme load 8%), the transesterification reaction was improved. However, when enzyme load was higher than 8% and substrates ratio was lower than 1:5, the biodiesel yield significantly decreased, which was ascribed to the free liquid lipase aggregation resulting in the decrease of enzyme activity per unit. There are also many reports about the increase in the enzyme load resulting in a decrease in the biodiesel yield [50]. 3.6. Optimum reaction variables and models verification The transesterification parameters were optimized as follows: substrate ratio 1:5 (mol/mol), enzyme load 10%, 30 C and 6.98 h. Under the optimized conditions, biodiesel yield was 98.8 ± 1.1%, which agreed with the predicted value (99.9%). The good consistency between predicted value and observed value indicated that the model was accurate. Compared with the immobilized lipases, similar high biodiesel yield can be obtained using the cheaper biocatalyst Lipozyme TL 100 L (140$/kg). And compared with chemical catalysts, the by-product glycerol obtained from the reaction system using Lipozyme TL 100 L as catalyst was better in color and residual inorganic salts. Therefore, compared with other catalysts, free lipase Lipozyme TL 100 L showed the potential alternative for biodiesel production. 3.7. Kinetic of the transesterification catalyzed by lipozyme TL 100 L The presence of excess free fatty acids can decrease biodiesel yield, which is due to more water formed by the esterification of free fatty acid with methanol. However, in this work, the free fatty acid content of PSTO was 4.2 ± 0.5%, and during the reaction progress, the free fatty acid content was maintained at 2.6 ± 0.9%. These results suggested that in this reaction system, the transesterification of the triacylglycerols in PTSO with methanol was the main reaction, and biodiesel was mainly produced by the transesterification. According to previous reports [51], the kinetic mechanism of the transesterification catalyzed by Lipozyme TL 100 L was accorded with Ping-Pong Bi-Bi mechanism. Therefore, the initial transesterification rates can be described as follows:
V0 ¼
Vmax ½A½B ½A½B þ ½BKA m þ ½A 1 þ K½AiA K m B
where
V0
and
Vmax
are
the
initial
(5)
and
the
maximum
transesterification rates, respectively. [A] and [B] are the concenm trations of methanol and PTSO, respectively. Km A and KB are the Michaelis-Meton constants. KiA is the inhibition constant of methanol. The relationship between the initial rate (V0) with the concentration of methanol ([A]) was shown in (Fig. 6). The initial transesterification rate (V0) significantly decreased when the concentration of methanol ([A]) was >3.3 mol/L (Fig. 6a), which was ascribed to the inhibition of methanol. Therefore, the kinetic constants Vmax and K’m of the transesterification catalyzed by Lipozyme TL 100 L were evaluated at low methanol concentrations (<3.3 mol/L). Keeping the mass of PTSO constant and varying the concentration of methanol from 0.668 mol/L to 3.342 mol/L, the transesterification can be deemed as a pseudo-first order reaction as follows.
V0 ¼
Vmax ½A ½A þ K ’m
(6)
where Vmax is the maximum methanol rate; [A] is the methanol concentration; K’m is the apparent Michaelis constant, respectively. Therefore, according to Lineweaver-burk double reciprocal method, the kinetic equation of the transesterification of PTSO catalyzed by free liquid Lipozyme TL 100 L can be obtained as follows:
1 2:83645 ¼ þ 0:40734 Vo ½A Consequently, Vmax and K’m of the transesterification catalyzed by liquid lipase Lipozyme TL100L were calculated as 6.9 103 mol/(L$min) and 2.55 101 mol/L, respectively. 4. Conclusions PTSO, as the novel resource, was successfully used to prepare biodiesel. Among the tested lipases, free liquid lipozyme TL100L from Thermomyces laguginosus showed the good resistance to methanol and catalysis preformance for the transesterification. Transesterification parameters were evaluated and optimized using RSM. The optimized conditions were as follows: substrate ratio 1:5 (mol/mol), enzyme load 10%, 30 C and 6.98 h. Under the optimal conditions, the maximum biodiesel yield (98.8 ± 1.1%) was achieved. The activation energy of biodiesel formation was 21.3 kJ/mol, and the kinetic constants K’m and Vmax were 2.55 101 mol/L and 6.9 103 mol/(L$min), respectively. These work showed that PTSO can be successfully used as novel potential raw material for biodiesel preparation and free liquid lipozyme TL100L as novel catalyst
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Fig. 5. 3D surface plots of biodiesel yield. (a) Reaction temperature and reaction time combined with the enzyme load and PTSO/methanol fixed at 9.7% and 1:4.6; (b) Reaction temperature and enzyme load for PTSO/methanol 1:4.6 (mol/mol) and 8.6 h; (c) Reaction temperature and substrate ratio for 9.2% enzyme load and 8.8 h; (d) Substrate ratio and reaction time with 9.2% enzyme load at 32.0 C; (e) Enzyme load and reaction time with PTSO/methanol 1:4.6(mol/mol) at 32.0 C. (f) Substrate ratio and enzyme load for 8.8 h at 32.0 C.
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References
Fig. 6. (a) The relationship between the initial transesterification rate (V0) with methanol concentration ([A]) at low methanol concentrations; (b) Lineweaver-burk. The reactions were conducted with 8% (v/v) Lipozyme TL 100 L load at 25 C.
for the transesterification.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (31771937) and the funding scheme for Young Teachers Cultivating Program in Henan University of Technology.
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Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L Shangde Sun*, Kaiyue Li Lipid Technology and Engineering, School of Food Science and Engineering, Henan University of Technology, Lianhua Road 100, Zhengzhou, 450001, Henan Province, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 April 2019 Received in revised form 29 October 2019 Accepted 2 November 2019 Available online xxx
Biodiesel is a green and sustainable energy, which is a fatty acid alkyl ester as an alternative to petroleum diesel. In the work, phoenix tree seed oil, one kind of undeveloped woody plant resource, was used as the novel raw material for biodiesel preparation. Several free liquid lipases were used as biocatalysts to catalyze the transesterification of phoenix tree seed oil (PTSO) with methanol. Effects of transesterification variables (enzyme load, substrate ratio, transesterification time and temperature) on biodiesel preparation were evaluated and optimized using RSM. Results showed that PTSO was a good alternative for biodiesel preparation. Among these tested free liquid lipases, lipozyme TL100L from Thermomyces laguginosus showed the best performance for the transesterification. Transesterification variables were optimized and the maximum biodiesel yield (98.8 ± 1.1%) was achieved under the optimal conditions (enzyme load 10%, transesterification temperature 30 C, substrate ratio (PTSO to methanol) 1:5 (mol/mol) and reaction time 6.98 h). The activation energy of biodiesel formation was 21.3 kJ/mol. Kinetic parameters K’m and Vmax were 2.55 101 mol/L and 6.9 103 mol/(L$min), respectively. © 2019 Elsevier Ltd. All rights reserved.
Keywords: Biodiesel Lipase Phoenix tree seed oil Transesterification Response surface methodology Kinetic
1. Introduction Biodiesel, as a green and sustainable energy, is consisted of mono-alkyl esters, mainly the methyl esters of fatty acids, which is non-toxic, biodegradable, renewable, and environmental [1e3]. The materials used for biodiesel preparation in the previous reports were mainly edible oils (for example, soybean oil, flaxseed oil and cottonseed oil) [4e8]. Compared with these edible oils, some nonedible vegetable oils and waste cooking oils have shown some advantages as the alternative resource [9e11]. Recently, some microbial oils and industry wastes have also been developed as the raw materials for biodiesel preparation [12e14]. Phoenix tree seed is one kind of undeveloped woody plant resource, which can be obtained from Phoenix tree grown by the road and in the courtyard in China [15,16]. In our previous report, we found the oil content of the seeds was 27.8 ± 0.3% [15]. In phoenix tree seed oil (PTSO), oleic acid (O, 22.2%), palmic acid (P, 17.43%) and linoleic acid (L, 30.2%) were the main fatty acids. However, no information using PTSO as raw material to prepare
* Corresponding author. E-mail addresses: [email protected] (S. Sun), [email protected] (K. Li).
biodiesel was available. In the previous reports about biodiesel preparation, different catalysts were used to catalyze the transesterification of oils with methanol, for example, acids [17,18], alkalis (sodium hydroxide, potassium hydroxide) [19,20] and enzymes (for example, immobilized lipases and free lipases from Pseudomonas aeruginosa, Yarrowia lipolytica and Rhizomucor miehei) [21e25]. Compared with acid and alkali, due to the mild reaction conditions, enzyme has shown good performance for the transesterification of oils [26,27]. And in these previous enzymatic transesterification of oils, immobilized enzymes were often used to prepare biodiesel [28,29]. Recently, free liquid lipases have attracted more attention as the alternative biocatalyst for biodiesel preparation [30e33]. However, the relative information using free lipase lipozyme TL100L as biocatalyst is enormously uncommon. In the work, PTSO, as the novel raw material, was used for biodiesel preparation. Several free liquid lipases were used as biocatalysts to catalyze the transesterification. Effects of transesterification variables (substrate ratio, enzyme load, time and temperature) on biodiesel preparation were evaluated. The interaction of transesterification variables were also investigated and optimized by response surface methodology (RSM). Reaction thermodynamic and kinetics were also analyzed.
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Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
2
S. Sun, K. Li / Renewable Energy xxx (xxxx) xxx
200 C at 2 C/min, and increased to 290 C at 20 C/min, and then increased to 320 C at 6 C/min and hold at 320 C for 1min, finally increased to 360 C at 20 C/min and hold at 360 C for 7min. Biodiesel yield was calculated as follows:
2. Materials and methods 2.1. Materials PTSO with 4.2 ± 0.5% free fatty acid content was prepared according to Soxhlet extraction method from phoenix tree seeds (Jiangsu, China). Free liquid lipases (lipase A from Candida sp. (CALA), Lipozyme from Thermomyces laguginosus (Lipozyme TL100L) and lipase B from Candida sp. (CALB) were provided by Novozymes A/S (Begsvaerd, Denmark).
Biodiesel yield ð%Þ ¼
mFAME 100% mTG þ mDG þ mMG þ mFAME þ mFA (1)
where m is the mass weight of product or reactants.
2.2. Enzymatic reaction of PTSO with methanol
2.4. RSM design
PTSO (2.50 g) was mixed with methanol (0.47 g) at 300 rpm and 30 C in 25-mL round-bottom flasks. Magnetic stirring bioreactor was used to mix reaction mixture and control the temperature for the reaction. The stirring speed was 300 rpm. When 0.20 g lipase was added into the mixture, the enzymatic transesterification was initiated. These standard conditions were used except when otherwise stated in the text. Samples (20 mL) were withdrawn using one 20 mL sample needle and dissolved in 3 mL n-hexane, and then dried using anhydrous sodium sulfate, and followed by centrifugation. Finally, the supernatant liquid was collected using one 2.5mL injector and filtered using one microfilter (0.45 mm) for GC analysis.
RSM with four factors and three levels was designed using BoxeBehnken in this work. The reaction variables and levels were transesterification temperature (20 C, 30 C, and 40 C), time (0.05, 7.00 and 13.95 h), enzyme load (1%, 8%, 15%; w/w) and substrate ratio (PTSO/methanol, 1:1, 5:1, 9:1 (mol/mol)) (Table 1).
2.3. Determination of biodiesel by GC
Y ¼ b0 þ
2.5. Statistical analysis From the RSM, 29 experiments were designed to evaluate the effect of four transesterification parameters on biodiesel yield. The quadratic polynomial equation between transesterification parameters with biodiesel yield was as follows: 4 X
bii X 2i þ
i1
Biodiesel samples were analyzed using GC (7890B) with a DB1ht capillary column (28 m 0.25 mm, 0.1 mm film thickness) and flame-ionization detector (FID). The flow of N2 was 4 mL/min. The temperatures of FID and injector were both 350 C. The column temperatures were firstly set at 170 C, secondly increased to
3 4 X X
bij Xi Xj
(2)
i1 jiþ1
where Y is biodiesel yield, Xi and Xj are the transesterification parameters, b0, bi, bii, and bij are the constant, linear, quadratic and cross term coefficients, respectively. To ensure the validity of the results, all experiments were
Table 1 RSM design and result of Lipozyme TL100L-catalyzed the transesterification of PTSO with methanol for biodiesel preparation. Treatmenta
Reaction time X1 (h)
Temperature X2 (oC)
Substrate ratio X3 (mol/mol)
Enzyme load X4 (%)
Biodiesel yield Y (%)
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29
7.00 (0) 7.00 (0) 0.05 (1) 13.95 (1) 7.00 (0) 0.05 (1) 7.00 (0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 7.00(0) 0.05(-1) 7.00(0) 13.95(1) 0.05(-1) 7.00(0) 7.00(0) 7.00(0) 13.95(1) 13.95(1) 0.05(-1) 7.00(0) 0.05(-1) 7.00(0) 13.95(1) 13.95(1)
30 (0) 30 (0) 20 (1) 30 (0) 40 (1) 30 (0) 20 (1) 20(-1) 40(1) 20(-1) 30(0) 30(0) 30(0) 20(-1) 30(0) 30(0) 40(1) 40(1) 40(1) 30(0) 40(1) 30(0) 30(0) 30(0) 30(0) 30(0) 30(0) 20(-1) 30(0)
5 (0) 1 (1) 5 (0) 9 (1) 5 (0) 5 (0) 1 (1) 9(1) 9(1) 5(0) 5(0) 1(-1) 5(1) 5(1) 1(-1) 9(1) 5(0) 5(0) 5(0) 9(1) 1(-1) 5(0) 5(0) 9(1) 5(0) 5(0) 5(0) 5(0) 1(-1)
8 (0) 1 (1) 8 (0) 8 (0) 1 (1) 15 (1) 8(0) 8(0) 8(0) 1(-1) 8(0) 15(1) 8(0) 15(1) 8(0) 1(-1) 8(0) 8(0) 15(1) 15(1) 8(0) 1(-1) 15(-1) 8(0) 8(0) 1(-1) 8(0) 8(0) 8(0)
95.7 ± 1.5 56.2 ± 1.0 76.3 ± 1.3 83.6 ± 1.2 18.6 ± 0.8 75.3 ± 1.4 68.6 ± 0.9 90.7 ± 1.1 55.9 ± 0.6 40.8 ± 1.7 95.9 ± 0.8 67.51 ± 1.0 94.0 ± 1.2 96.4 ± 0.8 34.8 ± 1.0 12.7 ± 0.9 96.7 ± 1.6 73.6 ± 1.3 95.7 ± 1.5 97.0 ± 3.0 67.4 ± 1.7 15.2 ± 0.7 96.0 ± 0.4 62.3 ± 1.0 94.0 ± 1.5 16.0 ± 0.9 97.1 ± 0.7 96.2 ± 1.7 48.2 ± 0.6
a
Numbers were run randomly.
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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performed at least in triplicate. The results were expressed as averages ± S.E.M. The significance of the difference was estimated using a two-way analysis of variance (ANOVA). Statistical significance was considered at p < 0.05. 3. Results and discussion 3.1. Liquid lipases screening The effect of free liquid lipases (Lipozyme TL100L, CALA and CALB) on the transesterification was shown in Fig. 1a. Among these lipases, the maximum biodiesel yield (80.0 ± 2.2%) was achieved using Lipozyme TL100L, which is about 6 times that of CALA (12.2 ± 1.5%) and CALB (14.7 ± 1.9%). These results were ascribed to the good resistance to methanol and phospholipid of Lipozyme TL100L. Compared with Lipozyme TL100L, the intra-protein
3
hydrogen bonding interactions of CALA and CALB associated with catalytic activity can be easily weakened in methanol, which resulted in the deactivation of CALA and CALB. Similar result that the hydrogen bond (H-bond) related with catalytic activity of CALB destroyed by methanol was also found [34]. In the previous report, lipase from P. cepacia also showed good methanol resistance at high methanol concentration [35]. Fig. 1b also shows that the initial reaction rates of the transesterification catalyzed by CALA and CALB (2.2e2.5 103 mol/(L$min)) were almost half of Lipozyme TL100L (4.5 103 mol/(L$min)). The time to achieve equilibrium using Lipozyme TL100L as catalysts was 7 h, which was very shorter than some immobilized lipases, for examples, immobilized Candida sp. 99e125 (24 h at 40 C) [36,] and immobilized Candida antarctica lipase (24 h at 50 C) [37]. These were ascribed to the lower mass transfer limitation of free liquid Lipozyme TL100L than the immobilized lipases. These can also be confirmed by the effect of stirring. When the stirring speed was greater than 300 rpm, no obvious change of biodiesel yield was found. These also indicated that external mass transfer limitation on the reaction can be eliminated. 3.2. Effect of temperature When the temperature increased from 10 C to 30 C, initial reaction rate increased from 4.0 103 mol/(L$min) to 8 103 mol/(L$min) and biodiesel yield also increased from 73.7 ± 1.7% to 95.3 ± 1.9% at 7 h (Fig. 2). The time to reach equilibrium also shortened from 7 h to 3 h. These results were due to the decrease of mass transfer limitation, and the increase of catalytic activity of enzyme and the number of activated molecules at high temperature. However, when the temperature varied from 30 C to 60 C, a remarkable decrease of biodiesel yield from 95.3 ± 1.9% to 17.3 ± 1.3% was found. The initial reaction rate also decreased from 8 103 mol/(L$min) to 1.3 103 mol/(L$min) and the time to reach equilibrium increased from 3 h to 12 h. The result was attributed to the deactivation of Lipozyme TL 100 L at high temperature. Similar deactivation at high temperature can also be found in other enzymatic reactions [22]. A linear relationship between initial reaction rate (lnVo) with temperature (1/T) (Y ¼ 2.56443Xþ3.58363,R2 ¼ 0.93118) was found (Fig. 2c), and Arrhenius equation of biodiesel formation can be obtained from the linear equation as follows.
LnVo ¼
2564:43 3:58363 ½A
(3)
Accordingly, the activation energy (Ea) of biodiesel formation by the transterification of PTSO using free liquid lipase Lipozyme TL 100 L as catalyst was calculated as 21.3 kJ/mol. Compared with the report of Guat et al. [38], the activation energy of Lipozyme TL IM is 22.2 kJ/mol, which is higher than free lipase Lipozyme TL 100 L.The lower activation energy means shorter equilibrium time. Fig. 2 also shows that, the optimal reaction temperature of free liquid Lipozyme TL 100 L was 30 C, which was lower than the immobilized Lipozyme TLIM (lipase from Thermomyces laguginosus) (40 C) [39]. Compared with the free liquid Lipozyme TL 100 L, high optimal reaction temperature was due to the protection of the immobilization of lipase from Thermomyces laguginosus. Compared with other lipases (for example, lipozyme TL IM [39e41] and Candida rogues lipase [42,43]), the optimal reaction temperature of free liquid Lipozyme TL 100 L (30 C) was low, which showed that Liposome TL 100 L was the best choice for biodiesel preparation. Fig. 1. (a) Effect of different liquid lipases (Lipozyme TL100L, CALA and CALB) on biodiesel yield; (b) Effect of different liquid lipases (Lipozyme TL100L, CALA and CALB) on initial reaction rate. The reactions were conducted with 1:5 (mol/mol) substrate ratio, 8% (v/v) lipase load with different enzymes at 40 C.
3.3. Effect of substrate ratio Methanol has significantly effect on enzyme activity. Biodiesel
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Fig. 2. (a) Effect of different reaction temperatures on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of different reaction temperatures on the initial reaction rates; (c) The relationship of initial reaction rate (lnV0) and reaction temperature (1/T). The reactions were conducted with 8% (v/v) Lipozyme TL 100 L lipase load and 1:5 (mol/mol) substrate ratio.
yield increased when substrate ratio (PTSO/methanol) changed from 1:4 to 1:5 (Fig. 3a). However, with further increase in methanol ratio from 1:5 to 1:15, biodiesel yield significantly decreased from 95.3 ± 1.9% to 55.7 ± 2.4%. Meanwhile, the initial reaction rate (Fig. 3b) also decreased from 6.9 103 mol/(L$min) to 2.2 103 mol/(L$min) and the time to reach equilibrium increased from 3 h to 7 h. The shortest time to reach equilibrium was 3 h. These were attributed to the deactivation of enzyme with high methanol concentrations [44]. Similar effect of methanol can also be found in other enzymatic reactions [45,46]. For example, the structure of Yarrowia lipolytica lipase was disrupted when methanol concentrations were higher than 30% [23]. The catalyst activity of Callera™ Trans L lipase from Thermomyces lanuginosus decreased when the molar ratio of triglyceride (degummed soybean oil) to methanol was 1:4.5 [47]. The catalyst activity of immobilized Candida rugosa lipase decreased when the substrate ratio of oil to methanol was lower than 1:4 [43]. Compared with these lipases, Lipozyme TL100L has high catalyst activity at 1:5 molar ratio of oil to methanol (Fig. 3). The methanol concentration was higher than those in the previous reports, which indicated that Lipozyme TL
100 L has high methanol tolerance in this reaction system. 3.4. Effect of enzyme load Fig. 4 shows that, biodiesel yield increased from 17.8 ± 0.7% to 95.2 ± 1.3% with enzyme load ranging from 1% to 10%, and the initial reaction rate also increased from 1.4 103 mol/(L$min) to 9 103 mol/(L$min) and the time to reach equilibrium also shortened from 7 h to 3 h. The maximum biodiesel yield (95.2 ± 1.3%) was obtained with 10% Lipozyme TL100L load at 7 h, which were ascribed to the presence of more effective catalytic sites at high enzyme load. However, when enzyme load increased from 10% to 15%, the initial reaction rate decreased from 9 103 mol/(L$min) to 12 103 mol/(L$min), however, the biodiesel yield also decreased from 95.2 ± 1.3% to 89.1 ± 2.1%, which could be due to the aggregation of Lipozyme TL100L and diffusion limitation of reaction system at high enzyme load. Similar results that, the catalyst efficiency per unit of enzyme decreased when excess enzyme was present in reaction system, can also be found in other enzymatic reactions [48,49]. After reaction was completed,
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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5
Fig. 3. (a) Effect of substrate ratio on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of substrate ratio on initial reaction rates. The reactions were conducted with 8% (v/v) Lipozyme TL 100 L load at 25 C.
the free lipase Lipozyme TL 100 L was separated and reused. However, biodiesel yield decreased by 45.5 ± 1.8% at the 2nd use of the lipase.
Fig. 4. (a) Effect of enzyme load on biodiesel yield in the transesterification of PTSO with methanol; (b) Effect of enzyme load on initial reaction rate. The reactions were conducted with 1:5 (mol/mol) substrates ratio at 30 C.
3.5. Model fitting To further improve the reaction efficiency for biodiesel preparation, the interaction of the transesterification variables were evaluated using RSM. For biodiesel preparation, the high coefficient (R2) of the model (0.9126) (Table 2) showed that the transesterification model was significant. Therefore, biodiesel yield can be satisfactorily explained by the following quadratic polynomial model: Biodieselyield (%) ¼ 3.97X1 þ 1.22X2 þ 13.57X3 þ 6.53X4 þ 0.01X1X2 þ 0.07X1X3 þ 0.11X1X4 - 0.21X2X3 þ 0.08X2X4 þ 0.65X3X4 - 0.31X21 - 0.02X22 - 1.17X23 - 0.53X24 -14.08 (4) The equation showed that X1, X2, X3, X4,X1X2, X1X3,X1X4,X2X4and X3X4 had positive effects on biodiesel yield. However, other parameters have negative effects on the transesterification.
Fig. 5 shows that the effect of reaction parameters on biodiesel yield decreased as substrates ratio > enzyme load > time > temperature. The effect of interaction of temperature, time, substrate ratio and enzyme load on biodiesel yield can be better evaluated using the 3D graphs. As shown in Fig. 5a, the optimal biodiesel yield appeared at 20 Ce30 C and 7 he10.48 h, and the effect of transesterification time on biodiesel yield was greater than reaction temperature. Similarly, the effects of enzyme load and substrate ratio on biodiesel yield were greater than that of reaction temperature (Fig. 5b and c). The optimal biodiesel yield appeared at 7.00 he10.48 h and substrate ratio from 1:3 to 1:7 with 11%e13% enzyme load (Fig. 5d and e). According to the RSM results, the effects of substrate ratio and enzyme load on biodiesel yield were higher than reaction time. The interaction (P ¼ 0.0087 < 0.05) of substrates ratio and enzyme load had a significant effect on biodiesel yield (Fig. 5f). The
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
6
S. Sun, K. Li / Renewable Energy xxx (xxxx) xxx
Table 2 ANOVA analysis for response surface model of biodiesel yield. Source
Sum of squares
Degree of freedom
Mean square
F value
Prob > F
Model X1 X2 X3 X4 X1X2 X1X3 X1X4 X2X3 X2X4 X3X4 Residual Lack of fit Pure error Total
20938.1 793.8 311.1 294.9 11310.5 2.6 15.6 115.6 282.2 115.6 1331.9 2005.6 1998.4 7.1 22943.7
14 1 1 1 1 1 1 1 1 1 1 14 10 4 28
1495.58 793.8 311.1 294.9 11310.5 2.6 15.6 115.6 282.2 115.6 1331.9 143.3 199.8 1.78 R2 ¼ 0.9126
10.44 5.54 2.17 2.06 78.95 0.018 0.11 0.81 1.97 0.81 9.30
<0.0001 0.0337 0.1627 0.1733 <0.0001 0.8956 0.7463 0.3843 0.1822 0.3843 0.0087
112.08
0.0002
optimal biodiesel yield appeared at 9%e15% enzyme load and 1:5 to 1:9 substrates ratio. When substrates ratio and enzyme load were lower than the optimum values (substrates ratio 1:5, enzyme load 8%), the transesterification reaction was improved. However, when enzyme load was higher than 8% and substrates ratio was lower than 1:5, the biodiesel yield significantly decreased, which was ascribed to the free liquid lipase aggregation resulting in the decrease of enzyme activity per unit. There are also many reports about the increase in the enzyme load resulting in a decrease in the biodiesel yield [50]. 3.6. Optimum reaction variables and models verification The transesterification parameters were optimized as follows: substrate ratio 1:5 (mol/mol), enzyme load 10%, 30 C and 6.98 h. Under the optimized conditions, biodiesel yield was 98.8 ± 1.1%, which agreed with the predicted value (99.9%). The good consistency between predicted value and observed value indicated that the model was accurate. Compared with the immobilized lipases, similar high biodiesel yield can be obtained using the cheaper biocatalyst Lipozyme TL 100 L (140$/kg). And compared with chemical catalysts, the by-product glycerol obtained from the reaction system using Lipozyme TL 100 L as catalyst was better in color and residual inorganic salts. Therefore, compared with other catalysts, free lipase Lipozyme TL 100 L showed the potential alternative for biodiesel production. 3.7. Kinetic of the transesterification catalyzed by lipozyme TL 100 L The presence of excess free fatty acids can decrease biodiesel yield, which is due to more water formed by the esterification of free fatty acid with methanol. However, in this work, the free fatty acid content of PSTO was 4.2 ± 0.5%, and during the reaction progress, the free fatty acid content was maintained at 2.6 ± 0.9%. These results suggested that in this reaction system, the transesterification of the triacylglycerols in PTSO with methanol was the main reaction, and biodiesel was mainly produced by the transesterification. According to previous reports [51], the kinetic mechanism of the transesterification catalyzed by Lipozyme TL 100 L was accorded with Ping-Pong Bi-Bi mechanism. Therefore, the initial transesterification rates can be described as follows:
V0 ¼
Vmax ½A½B ½A½B þ ½BKA m þ ½A 1 þ K½AiA K m B
where
V0
and
Vmax
are
the
initial
(5)
and
the
maximum
transesterification rates, respectively. [A] and [B] are the concenm trations of methanol and PTSO, respectively. Km A and KB are the Michaelis-Meton constants. KiA is the inhibition constant of methanol. The relationship between the initial rate (V0) with the concentration of methanol ([A]) was shown in (Fig. 6). The initial transesterification rate (V0) significantly decreased when the concentration of methanol ([A]) was >3.3 mol/L (Fig. 6a), which was ascribed to the inhibition of methanol. Therefore, the kinetic constants Vmax and K’m of the transesterification catalyzed by Lipozyme TL 100 L were evaluated at low methanol concentrations (<3.3 mol/L). Keeping the mass of PTSO constant and varying the concentration of methanol from 0.668 mol/L to 3.342 mol/L, the transesterification can be deemed as a pseudo-first order reaction as follows.
V0 ¼
Vmax ½A ½A þ K ’m
(6)
where Vmax is the maximum methanol rate; [A] is the methanol concentration; K’m is the apparent Michaelis constant, respectively. Therefore, according to Lineweaver-burk double reciprocal method, the kinetic equation of the transesterification of PTSO catalyzed by free liquid Lipozyme TL 100 L can be obtained as follows:
1 2:83645 ¼ þ 0:40734 Vo ½A Consequently, Vmax and K’m of the transesterification catalyzed by liquid lipase Lipozyme TL100L were calculated as 6.9 103 mol/(L$min) and 2.55 101 mol/L, respectively. 4. Conclusions PTSO, as the novel resource, was successfully used to prepare biodiesel. Among the tested lipases, free liquid lipozyme TL100L from Thermomyces laguginosus showed the good resistance to methanol and catalysis preformance for the transesterification. Transesterification parameters were evaluated and optimized using RSM. The optimized conditions were as follows: substrate ratio 1:5 (mol/mol), enzyme load 10%, 30 C and 6.98 h. Under the optimal conditions, the maximum biodiesel yield (98.8 ± 1.1%) was achieved. The activation energy of biodiesel formation was 21.3 kJ/mol, and the kinetic constants K’m and Vmax were 2.55 101 mol/L and 6.9 103 mol/(L$min), respectively. These work showed that PTSO can be successfully used as novel potential raw material for biodiesel preparation and free liquid lipozyme TL100L as novel catalyst
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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Fig. 5. 3D surface plots of biodiesel yield. (a) Reaction temperature and reaction time combined with the enzyme load and PTSO/methanol fixed at 9.7% and 1:4.6; (b) Reaction temperature and enzyme load for PTSO/methanol 1:4.6 (mol/mol) and 8.6 h; (c) Reaction temperature and substrate ratio for 9.2% enzyme load and 8.8 h; (d) Substrate ratio and reaction time with 9.2% enzyme load at 32.0 C; (e) Enzyme load and reaction time with PTSO/methanol 1:4.6(mol/mol) at 32.0 C. (f) Substrate ratio and enzyme load for 8.8 h at 32.0 C.
Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006
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References
Fig. 6. (a) The relationship between the initial transesterification rate (V0) with methanol concentration ([A]) at low methanol concentrations; (b) Lineweaver-burk. The reactions were conducted with 8% (v/v) Lipozyme TL 100 L load at 25 C.
for the transesterification.
Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (31771937) and the funding scheme for Young Teachers Cultivating Program in Henan University of Technology.
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Please cite this article as: S. Sun, K. Li, Biodiesel production from phoenix tree seed oil catalyzed by liquid lipozyme TL100L, Renewable Energy, https://doi.org/10.1016/j.renene.2019.11.006