Industrial Crops and Products 97 (2017) 218–223

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Industrial Crops and Products

journal homepage: www.elsevier.com/locate/indcrop

Enzymatic interesterification of crambe oil assisted by ultrasound

Gilmar Roberto Tavares a , José Eduardo Gonçalves 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
b
Programa de Mestrado em Tecnologias Limpas e Programa de Mestrado em Promoção 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

a r t i c l e i n f o a b s t r a c t

Article history: In this work, the production of fatty acid methyl esters (FAME) from crambe oil by enzyme interester-
Received 23 July 2016 ification assisted by ultrasound was investigated. The experiments evaluated the effect of temperature,
Received in revised form 5 October 2016 reaction time, methyl acetate (MA)/oil molar ratio, enzyme loading, catalyst reuse and influence of ultra-
Accepted 18 December 2016
sound. 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
Keywords:
to oil mass) gave the best yields. The use of methyl acetate promoted the enzyme stability throughout
Methyl acetate
successive cycles, with a decrease of only 10% and 8% in FAME yield and enzymatic activity. The results
Biodiesel
Crambe abyssinica H.
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 of biodiesel, with a low number of papers on the subject in the

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 excel-
lent fuel production (Wazilewski et al., 2013; Nadaletti et al., 2014)
with performance similar to that of mineral diesel and consider-
ably 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: camiladasilva.eq@gmail.com (C. da Silva).
literature.
In chemical routes conventionally used for the production of
biodiesel, transesterification of triglycerides with short chain alco-
hols 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 pro-
duction 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 tri-
acetin 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 tempera-
tures, which prevents the degradation of the products and reduces

http://dx.doi.org/10.1016/j.indcrop.2016.12.022
0926-6690/© 2016 Elsevier B.V. All rights reserved.
 G.R. Tavares et al. / Industrial Crops and Products 97 (2017) 218–223
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 cat-
alytic 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 trans-
fer between phases, and providing activation energy (Veljković
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 evalu-
ated 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
® internal standard and FAME yield was then calculated based on the
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 hydrox-
ide (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 com-
position 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
219
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 cat-
alyst 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 ther-
mostatic 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 accord-
ing 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 condi-
tions: 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
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 deter-
mine 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.
220 G.R. Tavares et al. / Industrial Crops and Products 97 (2017) 218–223
Table 1
Fatty acid composition of crambe oil.
Fatty acid Content (wt%)
Palmitic 2.24 ± 0.05
Palmitoleic 0.13 ± 0.01
Stearic 1.14 ± 0.04
Oleic 20.58 ± 0.63
Linoleic 4.71 ± 0.05
Linolenic 1.07 ± 0.10
Arachidonic 1.17 ± 0.02
Gadoleic 4.21 ± 0.02
Eicosadienoic 0.74 ± 0.01
Behenic 2.20 ± 0.01
Erucic 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 eru-
cic 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 triacylglyc-
erols 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 (rela-
tion 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 signifi-
cant 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 sub-
strate, 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).
100 c b b
b
b
c b
80
b a
a
FAME yield (wt%)
a
60 c
a
b
a
40
40 °C
50 °C
20
60 °C
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 ultra-
sound 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 temper-
ature 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 tem-
peratures 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, with-
out 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 accep-
tor/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.
 G.R. Tavares et al. / Industrial Crops and Products 97 (2017) 218–223 221

100 100
c
40
b
c 80
d
a d 30

Enzyme activity (U g )
b

-1
FAME yield (wt%)
80
FAME yield (wt%)

60

a 20
40

60
4 hours 10
20
6 hours

0 0
0 2 4 6
40
6 9 12 15
Reuse cycle

MA/oil molar ratio
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).
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.
100 b
b Moreover, with the use of 30 wt% of lipase it appears that
b b c the yield increase was not significant (p > 0.05) and using 40 wt%
c enzyme, notes a decrease of yield (p < 0.05). The observed decrease
a with increasing amount of enzyme can be explained by the fact
a
FAME yield (wt%)

80 that reactions with lipases occur in the substrates interface. With

MA/oil molar ratio
60
9
12
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 com-
parison 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 inter-
esterification of triolein with increasing enzyme concentration of
excess enzyme in the reaction medium can occur interface satura-
tion, 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, respec-
tively, 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 sur-
face 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
222 G.R. Tavares et al. / Industrial Crops and Products 97 (2017) 218–223
100
80
FAME yield (wt%)
60
40
20 without ultrasound
with ultrasound
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 activ-
ity 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 cavita-
tion. 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 pres-
sure, improving mass transfer and diffusion of substrate from the
catalytic site of the enzyme (Sutkar and Gogate, 2009; Veljković
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 conven-
tional 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.
4. Conclusion
The interesterification with methyl acetate assisted by ultra-
sound 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 sta-
bility 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.
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