Enzymatic interesterification of crambe oil assisted by ultrasound

Industrial Crops and Products 97 (2017) 218–223

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Enzymatic interesterification of crambe oil assisted by ultrasound Gilmar Roberto Tavares a , José Eduardo Gonc¸alves b , Wanderley Dantas dos Santos c , Camila da Silva d,∗ a

Departamento de Ciências Agronômicas, Universidade Estadual de Maringá (UEM), Estrada da Paca s/n, Umuarama, PR, 87500-000, Brazil Programa de Mestrado em Tecnologias Limpas e Programa de Mestrado em Promoc¸ão da Saúde, Centro Universitário de Maringá, Av. Guedner 1610, Maringa, PR, 87050-900, Brazil c Departamento de Bioquímica, Universidade Estadual de Maringá (UEM), Avenida Colombo 5790, Maringa, PR, 87020-900, Brazil d Departamento de Tecnologia, Universidade Estadual de Maringá (UEM), Avenida Ângelo Moreira da Fonseca 180, Umuarama, PR, 87506-370, Brazil b

a r t i c l e

i n f o

Article history: Received 23 July 2016 Received in revised form 5 October 2016 Accepted 18 December 2016 Keywords: Methyl acetate Biodiesel Crambe abyssinica H.

a b s t r a c t In this work, the production of fatty acid methyl esters (FAME) from crambe oil by enzyme interesterification assisted by ultrasound was investigated. The experiments evaluated the effect of temperature, reaction time, methyl acetate (MA)/oil molar ratio, enzyme loading, catalyst reuse and influence of ultrasound. The best results were obtained at 60 ◦ C, temperature at which the reaction reached equilibrium in 6 h. FAME yield was maximal at MA/oil molar ratio of 12 and the enzyme loading of 20 wt% (relative to oil mass) gave the best yields. The use of methyl acetate promoted the enzyme stability throughout successive cycles, with a decrease of only 10% and 8% in FAME yield and enzymatic activity. The results allow us to conclude that ultrasound reduced total reaction time and percentage of enzyme loading, when compared to the process without ultrasound. © 2016 Elsevier B.V. All rights reserved.

1. Introduction Biodiesel production is presented as sustainable alternative to petroleum diesel. For obtaining it should be prioritized non-food raw materials and with low cost. Crambe (Crambe abyssinica H.) is an interesting option, due to high oil content in its seeds (30–50%), short cycle (on average 90 days) and seed yield between 1000 and 1500 kg per hectare (Viana et al., 2013; Singh et al., 2014; Brandão et al., 2014; Prates et al., 2014). The crambe oil is predominantly formed by erucic acid (56–66%). The consumption of the this acid increases the level of cholesterol and lipidosis in heart tissues (Goswami et al., 2012), which makes the crambe unfit for human consumption (Wazilewski et al., 2013; Maciel et al., 2014). On the other hand, the erucic acid is stable at high temperatures and low melting point. The crambe oil still present high content of oleic acid and antioxidants allowing excellent fuel production (Wazilewski et al., 2013; Nadaletti et al., 2014) with performance similar to that of mineral diesel and considerably lower emissions of polluting gases (Rosa et al., 2014). Despite their qualities, crambe oil is still little explored for the production

∗ Corresponding author. E-mail address: [email protected] (C. da Silva). http://dx.doi.org/10.1016/j.indcrop.2016.12.022 0926-6690/© 2016 Elsevier B.V. All rights reserved.

of biodiesel, with a low number of papers on the subject in the literature. In chemical routes conventionally used for the production of biodiesel, transesterification of triglycerides with short chain alcohols such as methanol and ethanol leads to the formation of glycerol as byproduct. Thus, by classical methods, the increasing production of biodiesel lead to increased glycerol generation. As biodiesel production generates approximately 10% glycerol by volume, with the replacement of petroleum diesel per biodiesel, glycerol can make an economic and environmental liability (Leoneti et al., 2012). To mitigate this effect, it has been developed alternative route to the transesterification. The idea is to replace the alcohols by methyl acetate (MA) as acyl acceptor, route known as interesterification. In this new route, instead of producing glycerol, the reaction yields triacetin as coproduct. Triacetin has wide industrial application (Casas et al., 2013) and has no adverse effects on the quality of fuel (Saka and Isayama, 2009), being allowed its addition to biodiesel up to 10% by weight (Casas et al., 2011; Jung et al., 2012; Go et al., 2013). Among the catalysts used for interesterification of vegetable oils, immobilized enzymes show advantages, because are easily separated from the reaction medium resulting in greater purity of coproducts and reusability for several cycles without generating toxic waste (Ranganathan et al., 2008; Fjerbaek et al., 2009). The enzymes still allow the reactions are conducted at mild temperatures, which prevents the degradation of the products and reduces

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energy costs (Antczak et al., 2009). The use of methyl acetate have the additional advantage of not reduce the enzymatic activity in the early cycles, thus allowing more efficient re-use of enzymes (Huang and Yan, 2008; Ruzich and Bassi, 2010), which does not occur with the use of short chain alcohols (ethanol, methanol), because the glycerol can lead to inactivation of the enzyme, thereby decreasing its enzymatic activity (Jeong and Park, 2010). The use of immobilized catalysts may limit the mass transfer, since the supports may hinder the access of substrate to the catalytic site (Fjerbaek et al., 2009). The contact between the two phases is usually promoted by mechanical agitation. However, it has recently been shown that the application of ultrasound may offer advantages (Yu et al., 2010), because cavitation (formation, rise and implosion of bubbles in the reaction medium) generated by ultrasound accelerate chemical reactions by increasing mass transfer between phases, and providing activation energy (Veljkovic´ et al., 2012; Lerin et al., 2014). Cavitation causes localized increase in temperature on the border of the phases and mechanical energy which enhance mixing. The collapse of cavitation bubbles disrupts the boundary between phases, and promotes emulsification by ultrasonic jet. These effects provide increased reaction rates and obtaining high yields (Thank et al., 2010), reducing the need for large amounts of catalysts and the power consumption compared to the process with mechanical stirring (Chand et al., 2010). Based on the context described, in this work was investigated the enzymatic interesterification assisted by ultrasound of crambe oil, using methyl acetate as acyl acceptor. For this purpose we evaluated the effect of operational variables (temperature, time, enzyme loading and MA/oil molar ratio) in the FAME yield as well as reuse of the catalyst and the effect of ultrasound. 2. Material and methods 2.1. Materials Crambe oil donated by MS Foundation (cultivate Bright FMS), ® methyl acetate (Sigma Adrich, 99% purity) and lipase Novozym 435 were used in the experiments. In chromatographic analyzes were used standard chromatographic of methyl heptadecanoate (Sigma Adrich, >99% purity) and heptane as solvent (Anidrol). For the determination of enzyme activity and oil characterization were used: n-hexane (Panreac), lauric acid (Vetec), n-propyl alcohol (Panreac), acetone (Vetec), ethanol (Anidrol), sodium hydroxide (Anidrol), ethyl ether (Anidrol), phenolphthalein (Nuclear), methanol (BT Baker) and derivatising BF3 -methanol (Sigma Adrich). 2.2. Characterization of crambe oil The oil used in the reactions were characterized in terms of free fatty acids and fatty acid composition, using official methods recommended by American Oil Chemists’ Society (1990): 940.28 and Ce 2–66, respectively. After derivatization, the fatty acids composition was determined using the method described by Santos et al. (2015). The water content was determined using Karl Fischer titrator (Orion, AF8). 2.3. Experimental procedure The reactions were conducted in ultrasound bath with indirect contact (Unique Q 5.9/25 A), 165 W power and 25 kHz frequency, using round bottom flask, with a volume of 50 mL, as reactor positioned in the center of the ultrasonic bath. The reactor was connected to a condenser with water recirculation provided by thermostated bath (Marconi, model MA 184). The reactions were ® carried out using the enzyme Novozym 435, based on the works

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of Xu et al. (2003), Huang and Yan (2008) and Lei and Li (2015), who report higher yields with this enzyme. Prior to use, the enzyme catalyst was maintained at 40 ◦ C for 1 h in an oven with air circulation (Marconi, MA035) for its activation. At the same time, ultrasound bath was fired at 165 W and the reaction temperature, and the temperature being kept constant by means of a thermostated bath with water recirculation (Marconi, model MA 184). To check the effect of ultrasound in the process, reaction was performed on an orbital shaker (Marconi, MA 839/A) at 40 rpm. In each reaction, 1 g of oil was added to the flask together with methyl acetate (MA) and the enzyme, in amounts set for each experimental condition. After weighing the substrates and enzymes, the reactor was placed in ultrasonic bath and connected to the condenser coupled to thermostatic bath at 10 ◦ C. After the reaction time, removal of the enzymes was performed by filtration in quality paper with a diameter of 15 cm and weight of 80 g m−1 , and the excess solvent in the filtrate was evaporated via the evaporator route (Marconi, MA120) to constant weight and stored under refrigeration for conducting further analysis.

2.4. Determination of FAME yield To quantify the FAME content, samples were prepared according to the procedure reported by Silva et al. (2010). The analysis of samples was conducted in a gas chromatograph coupled to mass spectrum (Agilent) equipped with a capillary column Agilent HP-5MS (30 m × 0.250 mm × 0.25 ␮m), using the following conditions: injection 0.4 of ␮L in split mode 1:10, initial temperature of the column 120 ◦ C, maintained at this temperature for 5 min, increasing to 180 ◦ C at a rate of 15 ◦ C min−1 and for 240 ◦ C at a rate of 5 ◦ C min−1 , staying for five minutes. The flow of carrier gas, helium, was 1 mL min−1 . The temperature of ionization and quadrupole source were 230 and 150 ◦ C, respectively. Compounds were quantified upon analysis using methyl heptadecanoate as internal standard and FAME yield was then calculated based on the content of methyl esters in the analyzed sample and on the reaction stoichiometry (Tan et al., 2011; Maddikeri et al., 2013).

2.5. Reuse of biocatalyst and enzymatic activity After use, the enzymes were washed with 10 mL of methyl acetate (twice) and dried in oven at 40 ◦ C for 1 h. Recuperated enzyme was then kept in desiccator for 24 h prior to measurement of its activity and reutilization (Michelin et al., 2015). To determine the enzymatic activity was used the method described by Oliveira et al. (2006), wherein it consists in quantifying the lauric acid consumption in the esterification reaction between lauric acid and n-propyl alcohol, at 60 ◦ C with 5 wt% enzyme (in relation to the mass of substrate) kept under stirring for 40 min. To evaluate the reuse of the enzyme were adopted experimental conditions of MA/oil molar ratio of 12, 60 ◦ C, 30 wt% of enzyme (relative to mass of oil) and reaction time of 6 h. A total of five cycles were conducted and after each cycle the FAME yield and enzyme activity were evaluated.

2.6. Analysis of data The analyzes were performed in duplicate and data were sub® jected to ANOVA using Excel 2010 software and Tukey tests (using a 95% confidence interval), to evaluate differences among the media.

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Table 1 Fatty acid composition of crambe oil.

100 Content (wt%)

Palmitic Palmitoleic Stearic Oleic Linoleic Linolenic Arachidonic Gadoleic Eicosadienoic Behenic Erucic

2.24 ± 0.05 0.13 ± 0.01 1.14 ± 0.04 20.58 ± 0.63 4.71 ± 0.05 1.07 ± 0.10 1.17 ± 0.02 4.21 ± 0.02 0.74 ± 0.01 2.20 ± 0.01 61.82 ± 0.76

3. Results and discussion 3.1. Oil characterization Table 1 shows the fatty acid composition of the crambe oil obtained from the analysis by gas chromatography. According to Table 1, it was found that the crambe oil showed 61.82 wt% of erucic acid, which is in accordance with published studies that indicate contents from 58.5 to 63.77 wt% (Lalas et al., 2012; Onorevoli et al., 2014; Santos et al., 2015). Regarding the content of saturated and unsaturated fatty acids notes 6.88 and 93.13 wt% of these acids in crambe oil composition, respectively. Similar results were obtained by Santos et al. (2015) that found 6 and 94 wt% of saturated and unsaturated fatty acids, respectively. The oil showed 6.11 ± 0.20 wt% and 0.121 ± 0.001 wt% of free fatty acids and water, respectively. Analysis of these data indicates that this oil could not be intended for biodiesel production by the conventional method using homogeneous alkaline catalysts (Ma and Hanna, 1999). From the molar mass of the compounds identified in crambe oil (Table 1), the molar mass (MM) average of the triacylglycerols was estimated to 988.76 g mol−1 and the free fatty acids in 316.92 g mol−1 , and molar mass of crambe oil determined to be 875.42 g mol−1 .

3.2. Effect of temperature and reaction time To evaluate the effects of temperature and reaction time on enzymatic interesterification assisted by ultrasound reactions were carried out at MA/oil molar ratio of 12, with 30 wt% enzyme (relation to the oil mass), varying the temperature from 40 ◦ C to 60 ◦ C and it was considered reaction time of 2–10 h. The FAME yields are shown in Fig. 1, where it appears that the increased temperature favors the formation of esters. At 4 h of reaction, the yield increased from 54.66 wt% to 89.97 wt% with an increase in temperature from 40 to 60 ◦ C. This effect is also observed for times of 2–6 h. However, the results obtained in 8 and 10 h at 50 and 60 ◦ C showed no significant difference (p > 0.05), possibly because the reaction equilibrium was achieved. Yu et al. (2010) report that the increased temperature favors the frequency of collision between the enzyme molecules and substrate, which facilitates the formation of the enzyme-substrate complex and increased reaction rate. With increasing temperature also occurs a decrease in viscosity of the reaction medium, and thus, increases the mass transfer on the surface and inside of the enzyme particles (Otero et al., 2013; Cao et al., 2013). However, the gradual increase in temperature can cause denaturation of the enzyme, reducing the operational stability, catalytic reactivity and esters yield (Ruzich and Bassi, 2010; Lei and Li, 2015; Subhedar and Gogate, 2016).

c

b

80

a

b FAME yield (wt%)

Fatty acid

b b

b b

c

c

60

a

b a

40

a

a

40 °C 50 °C 60 °C

20

0 0

2

4

6

8

10

Reaction time (h) Fig. 1. Effect of temperature and reaction time on the FAME yield obtained using 30 wt% of catalyst (relative to oil mass) and MA/oil molar ratio of 12. Means followed by same letter (in the same time) did not differ statistically (p > 0.05).

The works of enzymatic interesterification with the lipase ® Novozym 435, without the use of ultrasound, report obtaining higher yields at temperatures of 40 ◦ C (Xu et al., 2003; Huang and Yan, 2008) and 50 ◦ C (Modi et al., 2007). In our research, the best results were obtained at 60 ◦ C. Increasing the temperature for ultrasound assisted processes is due to the effects of cavitation that providing higher yields (Yu et al., 2010). Batistella et al. (2012) reports obtaining higher yields of esters with increasing temperature from 40 to 70 ◦ C in the ultrasound assisted ethanolysis of ® soybean oil using the enzyme Novozym 435. Trentin et al. (2015) ◦ reports the temperature of 63 C as that provided maximum esters yield for ultrasound-assisted ethanolysis of soybean oil. The FAME yield increased with the reaction time for all temperatures studied, to reach equilibrium in 6 and 8 h at 50 and 60 ◦ C, respectively. Resulting in yields of 95.19 wt% at 50 ◦ C and 98.98 wt% at 60 ◦ C. Previous studies using enzymatic Interesterification, without ultrasound, report esters yields of 92, 98 and 90.1 wt% at 10 (Xu et al., 2003), 14 (Huang and Yan, 2008) and 25 h (Subhedar and Gogate, 2016), respectively. 3.3. Effect of MA/oil molar ratio Fig. 2 shows the effect of MA/oil molar ratio investigated at 4 and 6 h at 60 ◦ C and using 30 wt% of enzyme (in relation to mass oil). From the data presented in Fig. 2 can be seen that with increase of the MA/oil molar ratio of 6–12 increased FAME yield of 70.95–89.97 wt% and of 85.14–98.98% at 4 and 6 h, respectively. Fig. 2 In the interesterification reaction, three moles of methyl acetate are reacted with 1 mol of triacylglycerol resulting in 3 mol of esters and 1 mol of triacetin, indicating the stoichiometric MA/oil of 3. Since this is an equilibrium reaction, the excess MA shifts the reaction equilibrium favoring the formation of esters (Maddikeri et al., 2014; Subhedar and Gogate, 2016). Similar results are reported in the literature, where better yields in the enzymatic interesterification were found in the acyl acceptor/oil molar ratio of 12 (Xu et al., 2003; Du et al., 2004; Jeong and ® Park, 2010; Subhedar and Gogate, 2016). With the use of Novozym 435, Xu et al. (2003) report 60 and 90 wt% of esters yield for MA/oil molar ratio of 6 and 12, respectively, conducting the reaction at 40 ◦ C for 10 h using 30 wt% enzyme. Subheader and Gogate (2016) report yields of ∼41 and ∼90 wt% with use of MA/oil molar ratios of 6 and 12, respectively, at 40 ◦ C for 25 h, using 8 wt% of Lipozyme TL IM.

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a

40

c

80

d d FAME yield (wt%)

b

80

a

60

4 hours 6 hours

30 60 20 40

10

20

0

6

9

12

Fig. 2. Effect of MA/oil molar ratio on the FAME yield obtained at 60 ◦ C and using 30 wt% of catalyst (relative to oil mass). Means followed by same letter (in the same time) did not differ statistically (p > 0.05).

FAME yield (wt%)

100

b

b

b

b

c c

a a

MA/oil molar ratio 9 12

60

40 10

20

30

40

Enzyme loading (wt%) Fig. 3. Effect of enzyme loading on the FAME yield obtained at 60 ◦ C for 6 h of reaction. Means followed by same letter (in the same molar ratio) did not differ statistically (p > 0.05)..

However, excess reagent may causes excessive dilution of the reaction medium (Xu et al., 2003; Du et al., 2004; Maddikeri et al., 2013; Maddikeri et al., 2014; Subhedar and Gogate, 2016) and the possible substrate inhibition due to the large amount of methyl acetate used (Ruzich and Bassi, 2010), as can be seen in the comparison of results obtained in the molar ratios of 12 and 15, for the reaction times evaluated. 3.4. Effect of enzyme loading Fig. 3 shows the effect of the enzyme loading in the reaction investigated in 6 h of reaction at 60 ◦ C, MA/oil molar ratios of 9 and 12 and varying the enzyme loading between 10 and 40 wt% (relative to mass of oil). From the data presented in Fig. 3 it can be observed a significant increase (p < 0.05) on ester yield with the addition of enzyme amount of 10–20 wt% in molar ratios (MA/oil) 9 and 12. The increase in the amount of enzyme promotes contact between the substrate and the active sites of the catalyst (Trentin et al., 2015). Ruzich and Bassi (2010) reported higher esters yield from the interesterification of triolein with increasing enzyme concentration of

2

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Reuse cycle

15

MA/oil molar ratio

80

0 0

40

-1

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FAME yield (wt%)

100

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Enzyme activity (U g )

100

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Fig. 4. Effect of reuse recycle of the catalyst on the FAME yield (䊏 left vertical axis) and enzyme activity (䊉 right vertical axis) obtained at 60 ◦ C, using 30 wt% of catalyst (relative to oil mass) and MA/oil molar ratio of 12. Arrows refer to the meaning of the data sequence.

5–20 wt%, which was also observed by Jeong and Park (2010) for reaction between rapessed oil and methyl acetate. Moreover, with the use of 30 wt% of lipase it appears that the yield increase was not significant (p > 0.05) and using 40 wt% enzyme, notes a decrease of yield (p < 0.05). The observed decrease with increasing amount of enzyme can be explained by the fact that reactions with lipases occur in the substrates interface. With excess enzyme in the reaction medium can occur interface saturation, causing not all the enzyme active sites are still available for the substrate causing a resistance to mass transfer, becoming a factor limiting the reaction (Châabouni et al., 2006; Subhedar et al., 2015; Trentin et al., 2015). In this study, 20 wt% of enzyme afforded obtaining FAME yields of 95.11 and 98.20 wt% for molar ratios (MA/oil) of 9 and 12, respectively, at 6 h of reaction. Xu et al. (2003) reported obtaining ∼90 wt% of esters yield from soybean oil interesterified at 14 h of reaction using 30 wt% of catalyst. Modi et al. (2007) showed ∼95 wt% of esters yield for the interesterification of sunflower oil with ethyl acetate, using 10 wt% of catalyst in 12 h of reaction. Jeong and Park (2010) obtained as better conditions for the reaction between rapeseed oil and methyl acetate: MA/oil molar ratio of 12.44, 16.5 wt% of enzyme and 19.7 h reaction. In this condition, the authors report achieving 58 wt% esters content. The comparison of results from studies published in the literature reveals that the use of ultrasound increased yield, reduced reaction time and/or the enzyme loading. 3.5. Biocatalyst reuse The reuse of the catalyst was evaluated at 60 ◦ C using 30 wt% of enzyme (relative to mass of oil) and MA/oil molar ratio of 12. Fig. 4 shows the results obtained for the reuse of the enzyme for 5 cycles of 6 h each. It can be seen from the data presented in Fig. 4, that yield decreased only 10% while the enzymatic activity decreased 8% after ® 5 cycles evaluated. The high stability of Novozym 435 when used in the interesterification reaction is reported in several studies (Xu et al., 2003; Du et al., 2004; Lei and Li 2015). The production of biodiesel by the conventional method of transesterification generates glycerol which adheres to the surface of the enzyme and causes a decrease in catalyst activity (Du et al., 2004; Michelin et al., 2015). As the interesterification with methyl acetate, glycerol production does not occur, and this may encourage the reuse of the catalyst by more cycles with less loss in enzyme activity. This effect is evidenced when observing the results

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4. Conclusion

100

FAME yield (wt%)

80

60

40

without ultrasound with ultrasound

20

0 0

2

4

6

8

10

Reaction time (h) Fig. 5. Effect of ultrasound application on the FAME yield obtained at 60 ◦ C, using 30 wt% of catalyst (relative to oil mass) and MA/oil molar ratio of 12.

Batistella et al. (2012) that reports 40% of decrease in enzyme activity for the reaction of soybean oil with ethanol, after 7 cycles of 4 h each. ® The activity of Novozym 435 after successive reaction cycle with methyl acetate was reported in the work of Du et al. (2004) that not observe loss in the enzymatic activity of lipase after 100 cycle-reaction. Lei and Li (2015) also report that after use of the enzyme by 17 cycles of 8 h each, the esters yield decreases only 14%. Thus, the loss of enzyme activity obtained in this study are higher than shown in the literature, which is possibly due to the effect on the ultrasound waves in the characteristics of the enzyme, and the intensity and duration of exposure to ultrasonic irradiation can lead to inactivation of the enzyme due to cavitational collapses, the reported on the work of Batistella et al. (2012), Michelin et al. (2015) and Trentin et al. (2015).

3.6. Effect of ultrasound application The effect of ultrasound was investigated using 30 wt% of enzyme (relative to oil mass), MA/oil molar ratio of 12, 60 ◦ C in the range from 2 to 10 h reaction (Fig. 5). The use of ultrasound showed a significant increase (p < 0.05) of ∼38.7 and ∼8.3% in the FAME yield at 2 and 10 h of reaction, respectively. Although for 10 h the increase in yield is relatively small (<10%), the use of ultrasound accelerated the reaction rate and thus the reaction equilibrium was reached in less time compared to reaction without ultrasound (Fig. 5). The increase in yield can be attributed to the effects of cavitation. The ultrasonic waves cause increase the number of bubbles in the reaction medium increases the interaction between the enzyme and substrate (Yu et al., 2010; Lerin et al., 2014). The implosion of the cavitation bubbles produces local heating and increased pressure, improving mass transfer and diffusion of substrate from the catalytic site of the enzyme (Sutkar and Gogate, 2009; Veljkovic´ et al., 2012), having as effect the increasing of the reaction rate (Subhedar et al., 2015) Subhedar and Gogate (2016) report that the use of ultrasound reduces the reaction time considerably as compared to the conventional interesterification. In transesterification of the macauba oil with ethanol, Michelin et al. (2015) reported the increase of the esters yield of 62–75 wt% at 120 min reaction with application of ultrasound. Subhedar et al. (2015) obtained 95 wt% of FAME yield for the reaction assisted by ultrasound sunflower oil with methanol in 4 h, with the same conditions, without ultrasound, the authors report only 60 wt% yield after 24 h of reaction.

The interesterification with methyl acetate assisted by ultrasound was effective in FAME production from crambe oil using ® lipase Novozym 435 as catalyst. The use of ultrasound has reduced the enzyme loading to 20 wt% (relative to oil mass), increased the reaction rate and thus reduced the reaction time required to reach equilibrium. In the experimental range evaluated, it was observed that increasing MA/oil molar ratio to 12 and enzyme loading up to 20 wt% favored the production of esters. The best FAME yield observed was 98.25 wt% at 6 h and using MA/oil molar ratio of 12 and 20 wt% of enzyme. The catalyst reuse tests demonstrated stability for 30 h of use, with low loss of activity and production of esters. This work includes a contribution to the literature due to the lack of reports for production of crambe biodiesel and efficiency of interesterification assisted by ultrasound. Acknowledgement The authors are grateful to MS Foundation for the crambe oil donation and CNPq for financial support. References Antczak, M.S., Kubiak, A., Antczak, T., Bielecki, S., 2009. Enzymatic biodiesel synthesis–key factors affecting efficiency of processes. Renew. Energy 34, 1185–1194. Batistella, L., Lerin, L.A., Brugnerotto, P., Danielli, A.J., Trentin, C.M., Popiolski, A., Treichel, H., Oliveira, J.V., Oliveira, D., 2012. Ultrasound-assisted lipase-catalyzed transesterification of soybean oil in organic solvent system. Ultrason. Sonochem. 19, 452–458. Brandão, A.G., Silva, T.R.B., Henrique, L.A.V., Santos, J.S., Gonc¸alves, F.M., Kohatsu, D.S., Gonc¸alves Jr, A.C., 2014. Initial development of crambe due to sowing in different depths. Afr. J. Agric. Res. 9, 927–930. Cao, Y., Qi, S., Zhang, Y., Wang, X., Yang, B., Wang, Y., 2013. Synthesis of structured lipids by lipase-catalyzed interesterification of triacetin with camellia oil methyl esters and preliminary evaluation of their plasma lipid-lowering effect in mice. Molecules 18, 3733–3744. Casas, A., Ramos, M.J., Pérez, A., 2011. Kinetics of chemical interesterification of sunflower oil with methyl acetate for biodiesel and triacetin production. Chem. Eng. J. 171, 1324–1332. Casas, A., Ramos, M.J., Pérez, A., 2013. Methanol-enhanced chemical interesterification of sunflower oil with methyl acetate. Fuel 106, 869–872. Châabouni, M.K., Ghamghi, H., Bezzine, S., Rekik, A., Gargouri, Y., 2006. Production of flavour esters by immobilized Sthaphylococcus simulans lipase in a solvent-free system. Process Biochem. 41, 1692–1698. Chand, P., Chintareddy, V.R., Verkade, J.G., Grewell, D.A., 2010. Enhancing biodiesel production from soybean oil using ultrasonics. Energy Fuel 24, 2010–2015. Du, W., Xu, Y., Liu, D., Zeng, J., 2004. Comparative study on lipase-catalyzed transformation of soybean oil for biodiesel production with different acyl acceptors. J. Mol. Catal. B: Enzym. 30, 125–129. Fjerbaek, L., Christensen, K.V., Norddahl, B.A., 2009. Review of the current state of biodiesel production using enzymatic transesterification. Biotechnol. Bioeng. 102, 1298–1302. Go, A., Lee, Y., Kim, Y.H., Park, S., Choi, J., Lee, J., Han, S.O., Kim, S.W., Park, C., 2013. Enzymatic coproduction of biodiesel and glycerol carbonate from soybean oil in solvent-free system. Enzyme Microb. Technol. 53, 154–158. Goswami, D., Basu, J.K., De, S., 2012. Optimal hydrolysis of mustard oil to erucic acid: a biocatalytic approach. Chem. Eng. J. 182, 542–548. Huang, Y., Yan, Y., 2008. Lipase-catalyzed biodiesel production with methyl acetate as acyl acceptor. Z. Naturforsch. C 63c, 297–302. Jeong, G.T., Park, D.H., 2010. Synthesis of rapeseed biodiesel using short-chained alkyl acetates as acyl acceptor. Appl. Biochem. Biotechnol. 161, 195–208. Jung, H., Lee, Y., Kim, D., Han, S.O., Wook, S., Lee, K.J., Kim, Y.H., Park, C., 2012. Enzymatic production of glycerol carbonate from by-product after biodiesel manufacturing process. Enzyme Microb. Technol. 51, 143–147. Lalas, S., Gortzi, O., Athanasiadis, V., Dourtoglou, E., Dourtoglou, V., 2012. Full characterisation of Crambe abyssinica Hochst seed oil. J. Am. Oil Chem. Soc. 89, 2253–2258. Lei, Q., Li, T., 2015. Functional monoesters of jojoba oil can be produced by enzymatic interesterification: reaction analysis and structural characterization. Eur. J. Lipid Sci. Technol. 17, 630–636. Leoneti, A.B., Leoneti, V.A., Oliveira, S.V.W.B., 2012. Glycerol as a by-product of biodiesel production in Brazil: alternatives for the use of unrefined glycerol. Renew. Energy 45, 138–145. Lerin, L.A., Loss, R.A., Remonatto, D., Zenevicz, M.C., Balen, M., Netto, V.O., Ninow, J.L., Trentin, C.M., Oliveira, J.V., Oliveira, D., 2014. Areview on lipase-catalyzed reactions in ultrasound-assisted systems. Bioproc. Biosyst. Eng. 37, 2381–2394.

G.R. Tavares et al. / Industrial Crops and Products 97 (2017) 218–223 Ma, F., Hanna, M., 1999. Biodiesel production: a review. Bioresour. Technol. 70, 1–15. Maciel, A.M., Ming, C.C., Ribeiro, A.P.B., Silva, R.C., Gioielli, L.A., Gonc¸alves, L.A.G., 2014. Physicochemical properties of interesterified blends of fully hydrogenated Crambe abyssinica oil and soybean oil. J. Am. Oil Chem. Soc. 91, 111–123. Maddikeri, G.L., Pandit, A.B., Gogate, P.R., 2013. Ultrasound assisted interesterification of waste cooking oil and methyl acetate for biodiesel and triacetin production. Fuel Process. Technol. 116, 241–249. Maddikeri, G.L., Gogate, P.R., Pandit, A.B., 2014. Intensified synthesis of biodiesel using hydrodynamic cavitation reactors based on the interesterification of waste cooking oil. Fuel 137, 285–292. Michelin, S., Penha, F.M., Sychoski, M.M., Scherer, R.P., Treichel, H., Valério, A., Di Luccio, M., Oliveira, D., Oliveira, J.V., 2015. Kinetics of ultrasound-assisted enzymatic biodiesel production from macauba coconut oil. Renew. Energy 76, 388–393. Modi, M.K., Reddy, J.R.C., Rao, B.V.S.K., Prasad, R.B.N., 2007. Lipase-mediated conversion of vegetable oils into biodiesel using ethyl acetate as acyl acceptor. Bioresour. Technol. 98, 1260–1264. Nadaletti, W.C., Bariccatti, R.A., Alves, H.J., Souza, S.N.M., Antonelli, J., Cremonez, P.A., Belli Filho, P., Mari-Junior, A., 2014. Factorial planning and surface response methodology in oxidative stability evaluation of biodiesel blends from soybean crambe, babassu and swine fat. J. Food Agric. Environ. 12, 1030–1032. Oliveira, D., Feihrmann, A.C., Rubira, A.F., Kunita, M.H., Dariva, C., Oliveira, J.V., 2006. Assessment of two immobilized lipases activity treated in compressed fluids. J. Supercrit. Fluid 38, 373–382. Onorevoli, B., Machado, M.E., Dariva, C., Franceschi, E., Krause, L.C., Jacques, R.A., Caramão, E.B., 2014. A one-dimensional and comprehensive two-dimensional gas chromatography study of the oil and the bio-oil of the residual cakes from the seeds of Crambe abyssinica. Ind. Crop Prod. 52, 8–16. Otero, C., Márquez, P., Criado, M., Hernández-Martín, E., 2013. Enzymatic interesterification between pine seed oil and a hydrogenated fat to prepare semi-solid fats rich in pinolenic acid and other polyunsaturated fatty acids. J. Am. Oil Chem. Soc. 90, 81–90. Prates, F.B.S., Genuncio, G.C., Ferrari, A.C., Nascimento, E.C., Alvez, G.Z., Palermo, D.P., Zonta, E., 2014. Acúmulo de nutrientes e produtividade de crambe em func¸ão da fertilizac¸ão com torta de mamona e serpentinito. Cienc Rural 44, 810–816. Ranganathan, S.V., Narasimhan, S.L., Muthukumar, K., 2008. An overview of enzymatic production of biodiesel. Bioresour. Technol. 99, 3975–3981. Rosa, H.A., Wazilewski, W.T., Secco, D., Chaves, L.I., Veloso, G., Souza, S.N.M., Silva, M.J., 2014. Biodiesel produced from crambe oil in Brazil A study of performance and emissions in a diesel cycle engine generator. Renew. Sust. Energy Rev. 38, 651–655. Ruzich, N.I., Bassi, A.S., 2010. Investigation of enzymatic biodiesel production using ionic liquid as a co-solvent. Can. J. Chem. Eng. 88, 277–282.

223

Saka, S., Isayama, Y., 2009. A new process for catalyst-free production of biodiesel using supercritical methyl acetate. Fuel 88, 1307–1313. Santos, K.A., Bariccatti, R.A., Filho, L.C., Schneider, R., Palú, F., Silva, C., Silva, E.A., 2015. Extraction of crambe seed oil using subcritical propane: kinetics: charactetization and modeling. J. Supercrit. Fluid 104, 54–61. Silva, C., Castilhos, F., Oliveira, J.V., Filho, L.C., 2010. Continuous production of soybean biodiesel with compressed ethanol in a microtube reactor. Fuel Process. Technol. 91, 1274–1281. Singh, B.K., Bala, M., Rai, P.K., 2014. Fatty acid composition and seed meal characteristics of Brassica and allied genera. Natl. Acad. Sci. Lett. 37, 219–226. Subhedar, P.B., Gogate, P.R., 2016. Ultrasound assisted intensification of biodiesel production using enzymatic interesterification. Ultrason. Sonochem. 29, 67–75. Subhedar, P.B., Botelho, C., Ribeiro, A., Castro, R., Pereira, M.A., Gogate, P.R., Paulo, A.C., 2015. Ultrasound intensification suppresses the need of methanol excess during the biodiesel production with Lipozyme TL-IM. Ultrason. Sonochem. 27, 530–535. Sutkar, V.S., Gogate, P.R., 2009. Design aspects of sonochemical reactors: techniques for understanding cavitational activity distribution and effect of operating parameters. Chem. Eng. J. 55, 26–36. Tan, K.T., Lee, K.T., Mohamed, A.R., 2011. Prospects of non-catalytic supercritical methyl acetate process in biodiesel production. Fuel Process. Technol. 92, 1905–1909. Thank, L.T., Okitsu, K., Sadanaga, Y., Takenaka, N., Maeda, Y., Bandow, H., 2010. Ultrasound-assisted production of biodiesel fuel from vegetable oils in a small scale circulation process. Bioresour. Technol. 101, 639–645. Trentin, C.M., Popiolki, A.S., Batistella, L., Rosa, C.D., Treichel, H., Oliveira, D., Oliveira, J.V., 2015. Enzyme-catalyzed production of biodiesel by ultrasound-assisted ethanolysis of soybean oil in solvent-free system. Bioprocess. Eng. 38, 437–448. ´ V.B., Avramovic, J.M., Stamenkovic, O.S., 2012. Biodiesel production by Veljkovic, ultrasound-assisted transesterification: state of the art and the perspectives. Renew. Sust. Energy Rev. 16, 1193–1209. Viana, O.H., Borsoi, A., Santos, R.F., Sanderson, K., 2013. Productivity in relation to spatial arrangement and fertilization in the culture of Crambe abyssinica Hochst. J. Food Agric. Environ. 11, 721–723. Walker, R.E., 1990. Official Methods and Recommended Practices of the American Oil Chemists’ Society, 4th Ed., Champaign. Wazilewski, W.T., Bariccatti, R.A., Martins, G.I., Secco, D., Souza, S.M.N., Rosa, H.A., Chaves, L.I., 2013. Study of the methyl crambe (Crambe abyssinica Hochst) and soybean biodiesel oxidative stability. Ind. Crop. Prod. 43, 207–212. Xu, Y., Du, W., Liu, D., Zeng, J., 2003. A novel enzymatic route for biodiesel production from renewable oils in a solvent-free medium. Biotechnol. Lett. 25, 1239–1241. Yu, D., Tian, L., Wu, H., Wang, S., Wang, Y., Ma, D., Fang, X., 2010. Ultrasonic irradiation with vibration for biodiesel production from soybean oil by Novozym 435. Process Biochem. 45, 519–525.