Microporous and Mesoporous Materials 268 (2018) 39–45

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Microporous and Mesoporous Materials

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

Using factorial experimental design to optimize biocatalytic biodiesel T

production from Mucor Miehei Lipase immobilized onto ordered mesoporous
materials
C. Cartereta,∗, J. Jacobyb, J.L. Blinb
a
Université de Lorraine/CNRS, LCPME, UMR7564, 54600 Villers-lès-Nancy, France
b
Université de Lorraine/CNRS, L2CM, UMR7053, F-54506 Vandœuvre-lès-Nancy Cedex, France

A R T I C LE I N FO A B S T R A C T

Keywords: A supported biocatalyst was prepared by immobilization of Mucor miehei lipase onto mesoporous silica materials.
Mesoporous silica This material was used for the synthesis of biodiesel through the transesterification of rapeseed oil with me-
Lipase thanol. The process of biodiesel production was optimized by application of the factorial design methodology.
Supported biocatalyst Methanol to oil molar ratio, water addition, reaction temperature and biocatalyst content were chosen as the
Transesterification
variables and the response selected was the yield of esterification. Oil/methanol ratio was found to have the
Design of experiments
strongest influence on conversion, a low ratio is more favorable. To increase the enzyme activity water has to be
added. However excess of water favors the hydrolysis reaction. The temperature has a slight influence. The
optimal conditions for the transesterification are: reaction temperature of 24 °C, alcohol/oil molar ratio of 1:1,
and water content of 2.5% w/w. Under these reaction conditions the methanol is totally consumed and around a
third of the triglycerides are converted. Nearly 100% of the oil was converted to methyl esters with incremental
additions of methanol (three increments of methanol at regular time).

1. Introduction
Enzymes are catalysts of biological origin that have an important
catalytic power and a high degree of specificity. Most of the enzymes
are proteins found in living organisms with complex structures.
Enzymes offer a distinct advantage over other kinds of catalysts due to
their specificity: chemo-, regio- and stereo-selectivity, the mild condi-
tions for their reactions, and their eco-friendliness. Their catalytic ac-
tivity depends on the integrity of their native protein conformation. If
an enzyme is denatured or dissociated into subunits, its catalytic ac-
tivity is lost. However these biocatalysts present some drawbacks.
Indeed, free enzymes are labile and are not always sufficiently stable
under operational conditions and one time usage, as catalyst is costly.
In addition the separation of enzyme from solutions is very difficult
which makes the recycling almost impossible. To overcome these
drawbacks, one way consists in immobilization onto supports [1–4].
Thanks to their properties such as high specific surface area, high pore
volume and tunable pore size, mesoporous silica are materials are ex-
cellent candidates to be use for the preparation of supported biocata-
lysts [5–9]. In addition, silica, especially the one prepared by sol-gel
chemistry, is also known to be safe, not only for the environment, but
also for the human body within a certain dose [10–12]. SBA-15 is of
peculiar interest since it possesses a regular hexagonal array of pores
with uniform diameter, a very high specific surface area and an im-
portant pore volume [13]. With pore diameters of 8–10 nm, SBA-15 is
much more suitable for enzyme immobilization [14]. In addition the
thick silica walls make the final material stable and resistant. These
thick silica walls are a specificity of the material and involve higher
mechanical resistance. The immobilization can be achieved by different
methods such as entrapment, cross linking, chemisorption or physi-
sorption [9,15–18]. The latest process, where only weak bonds such as
hydrogen bonds or Van Der Waals bonds are used is the simplest
technique since it requires the dipping of the carrier in an enzyme so-
lution at the appropriate concentration and for the appropriate time.
After immobilization improvements in the activity and stability of the
immobilized enzyme can be observed [19,20].
Among the various enzymes, which can be immobilized onto porous
supports, lipases are in particular interest. Lipases play a crucial role in
metabolism and fat digestion. They are recognized as one of the most
important group of enzymes in biotechnology, with applications in
food, detergent, pharmaceutical, leather, textile, cosmetic and paper
industries [14,21]. In our group we are in particular interested in the

∗
Corresponding author.
E-mail address: cedric.carteret@univ-lorraine.fr (C. Carteret).

https://doi.org/10.1016/j.micromeso.2018.04.004
Received 18 December 2017; Received in revised form 18 March 2018; Accepted 2 April 2018
Available online 03 April 2018
1387-1811/ © 2018 Elsevier Inc. All rights reserved.
C. Carteret et al.
lipase originating from the Mucor miehei (Mm-L) which is extracted
from the Aspergillus oryzae [22,23]. Because of its stability this lipase is
excellent candidate for esterification reactions and Mm-L is involved in
the development of industrial biocatalysts [24], in particular Mm-L is
used for transesterification reaction involved in biodiesel production
from vegetable oils [25–29]. The transesterification yield will depend
on different parameters such as the alcohol/oil molar ratio, the tem-
perature, the water content in the mixture and so on [25,30,31]. The
best way to optimize each parameter taking into account the possible
interactions between them is the use of experimental design metho-
dology. Moreover, it allows describing well a system from a lower
number of experiments than a systematic investigation.
These experimental designs have been used to optimize the trans-
esterification reactions catalyzed by free lipase [32–35] but they have
been barely considered for supported biocatalysts [36,37]. For ex-
ample, using this approach Nguyen et al. [37] have determined the
optimal reaction conditions for the biodiesel production from the
commercial Novozym 435. They have shown that under these optimal
conditions a 96.18% maximum biodiesel yield can be reached at 26 °C.
In a previous work we have shown that mesoporous silica is a suitable
support for the physisorption of Mm-L [22]. The obtained supported
biocatalysts can effectively be used to catalyze the transesterification of
rapeseed oil with methanol, giving up to 76% yield. Nevertheless the
different parameters of the reaction have not been optimized. Here we
have investigated the influence of synthesis parameters (the methanol/
oil molar ratio, catalyst weight, temperature, water) on the transes-
terification yield under the experimental design methodology. The
statistical analysis allows links to be revealed, which would otherwise
have been difficult to observe, through analyzing both the main effect
of a factor and the interaction effects between the factors on the me-
thanol conversion rate. This study is of particular interest since sys-
tematical investigation of each parameter that might influence the re-
action performance is compulsory before any large scale manipulation.
2. Materials and methods
2.1. Support preparation
SBA-15 type material with a pore size of 8.5 nm has been synthe-
sized from the triblock copolymer Pluronic P123 (EO)20(PO)70 (EO)20
(Aldrich) according to published procedure reported in literature [38].
2.2. Mucor Miehei adsorption
Mm-L purchased from Aldrich was used as received without any
purification. The quantity of proteins contained in the commercial
powder, determined by the Bradford assay is around 2.2%. The activity
of the Mucor Miehei Lipase has been determined in mild reaction con-
ditions by the hydrolysis of triolein at pH 8 and 40 °C. The activity of
Mm-L is 1.39 U/mg, where 1 U corresponds to the amount of enzyme
which releases 1 μmol of oleic acid per minute at pH 8 and 40 °C.
Initially, a 0.2 mol L−1 Tris solution (pH = 10.8) was prepared.
Then the pH of the solution was adjusted with hydrochloric acid
(1 mol L−1) to 6. Previous results showed that when the adsorption is
performed at pH = 3 or 10, the activity of the lipase is quenched
whereas after absorbing the lipase at pH = 6, the supported biocatalyst
can effectively be used for transesterification of rapeseed oil with me-
thanol [15]. After that the enzyme was dissolved in 4 mL of the solu-
tion. The loading of enzyme was varied from 0 to 40 mg per mL of
solution. After homogenization during 30 min, 250 mg of the silica
support was added to the enzyme solution. The mixture was shaken at
room temperature until equilibrium time. The retrieved solid was wa-
shed 3 times with 5 mL of the solution before it was left to air-dry at
room temperature.
40
Microporous and Mesoporous Materials 268 (2018) 39–45
2.3. Characterization
SAXS measurements were carried out using SAXSess mc2 (Anton
Paar) apparatus. It is attached to a ID 3003 laboratory X-Ray generator
(General Electric), equipped with a sealed X-ray tube (PANalytical, λCu
(Kα) = 0.1542 nm, P = 3.3 kW). Nitrogen adsorption – desorption iso-
therms were obtained at −196 °C with a volumetric adsorption ana-
lyzer TRISTAR 3000 manufactured by Micromeritics. The samples were
degassed under vacuum for several hours at room temperature before
nitrogen adsorption measurements. The specific surface area was de-
termined by the BET (Brunauer, Emmett, Teller) method (molecular
cross sectional area = 0.162 nm2) and the pore diameter and the pore
size distribution by the BJH (Barret, Joyner, Halenda) method [39]. The
infrared spectra were recorded on a FTIR spectrometer Nicolet 8700,
equipped with a KBr beam splitter and a DTGS detector. The spectra in
diffuse reflectance mode were collected using a Harrick Praying
Mantis™ equipment. To perform the analysis, the mesoporous silica
powder (5 wt%) was mixed with KBr. Reflectances Rs of the sample and
Rr of pure KBr, used as a non-absorbing reference powder, were mea-
sured under the same conditions. The relative reflectance is defined as
R = Rs/Rr. The spectra are shown in pseudo-absorbance (−log R)
mode. The quantity of adsorbed proteins has been determined from the
infrared analyses. To do that first, in order to obtain a calibration line,
various amounts of enzyme (from 0.1 to 1 mg) were dispersed in 1 mg
of pure silica mesoporous materials. The obtained mixtures were then
diluted in KBr (5 wt%). Spectra were recorded and the ratio between
the area of the bands characteristics of the peptide group and the area
of the bands corresponding to the internal standard was plotted versus
the amount of enzyme. Then, the enzymes-loaded silica materials were
analyzed and the quantity of adsorbed proteins has been evaluated. It
should be outlined that since the Mm-L has been used as received
without purification, only the total protein content can be evaluated.
2.4. Transesterification reaction
Different samples are prepared by mixing into different closed glass
vials rapeseed oil with methanol at different alcohol to oil molar ratios.
At this stage different wt.% of water are added. The reaction was in-
itiated by the addition of the supported biocatalyst, i.e. the mesoporous
materials containing the immobilized Mucor miehei. The mixtures were
incubated at different temperatures with a constant shaking at 100 rpm.
The samples were centrifuged during 15 min at 5000 rpm. This allows
separating reaction products, i.e. the methyl esters from the supported
biocatalyst and glycerol, which is a by-product. The products are then
analyzed by GC-MS that allows the identification of the obtained pro-
ducts. More details on the protocol are reported in a previous paper
[22].
2.5. Design of experiments
The optimization of the variables affecting the synthesis of Methyl
Ester (ME) was carried out following design of experiments metho-
dology. The response measured, Y, was methanol conversion to ME
after 72 h. A 24 full factorial design with four center-points was carried
out to study the main effects and interactions between factors pre-
viously selected by screening designs. The factors chosen were me-
thanol to rapeseed oil molar ratio, temperature, wt% of water, and
catalyst amount. Stirring and oil volume were fixed at 100 rpm and
1.5 mL, respectively.
The levels of the factors were chosen according to preliminary re-
sults. Indeed, the data obtained showed that the methanol to oil molar
ratio is a main parameter and that a ratio greater than 3 leads to almost
zero yield. This is due to denaturation of the lipase by methanol. To
increase the enzyme activity water has to be added. However excess of
water favors the hydrolysis reaction. The temperature has not to exceed
44 °C.
C. Carteret et al. Microporous and Mesoporous Materials 268 (2018) 39–45

The experimental design and analysis of results were carried out Table 1
using MINITAB software package. A linear mathematical model to Values of the pore diameter (∅), the specific surface area (SBET) and the pore
predict the effect of the selected factors on the Methyl Ester Yield was volume (Vp) of SBA-15 after its stay during 210 min in the tris solution at pH 6
generated: with different contents of Mm-Lipase.
Mm-L (mg/L) ∅ (nm) SBET (m2/g) Vp (cm3/g)
y = β0 + ∑ βi Xi + ∑ ∑ βij Xi Xj
i i j>i 0 8.2 417 0.65
2 8.1 377 0.59
where y is the response, xi and xj represent the coded independent 7 8.1 356 0.55
variables, β0 is the constant, βi is the linear term coefficient, βij is the 10 8.3 340 0.53
cross-term or interaction coefficient. Main effects and interaction effects
correspond to the linear and cross term of the model; they are briefly
explained here. The main effect of a factor corresponds to the change of
the mean of the response from one level of the factor to another level
whatever the level of the other factors. If the change in the mean of the
response from one level of a factor to another level depends on the level
of another factor, the two factors are said to have interaction effects.
The statistical significance of the model (and the influence of the
parameters) was performed by the analysis of variance (ANOVA). The
center points make it possible to estimate the experimental error and to
test the linear model associated with this full factorial design. The
yields obtained for these points show good repeatability.

3. Results and discussion

3.1. Biocatalysts characterization

3.1.1. Lipase immobilization

Before the Mm-L adsorption, we have evaluated the stability of the
SBA-15 in the same conditions used for the lipase immobilization. After
immersion during 210 min in the solution at pH = 6, the Small Angle X-
ray Scattering (SAXS) pattern of the silica support exhibits a sharp peak
at 10.0 nm, two peaks at 5.7 and 5.0 nm (Fig. 1), which indicate a
hexagonal organization of the channels in this material. The specific
surface area, the pore volume and the pore diameter values are given in
Table 1. The mesopore ordering of the matrix is preserved.
Upon the Mm-L adsorption when the enzyme concentration is raised
up to 20 mg/mL, the hexagonal structure of the SBA-15 support is
maintained (Fig. 1) and in the same time a decrease of both the specific
surface area and the pore volume is noted, while the pore diameter
remains almost unchanged up to 10 mg of lipase per mL (Table 1).
Going from 0 to 10 mg of lipase per mL in the starting solution, the
values of the specific surface area and the pore volume slowly decrease
from 417 to 340 m2/g and from 0.65 to 0.53 cm3/g, respectively.
Fig. 2. Infrared spectra of SBA-15 after 210 min in the solution (pH = 6) at
different concentrations of lipase (a) 0; (b) 2; (c) 7; (d) 10; (e) 20 mg/mL.
Taking into account the changes of the textural properties in the pre-
sence of the lipase as well as the spherical molecular diameter of Mucor
Miehei, which is around 3.5 nm [40], we can conclude that the enzyme
enters into the mesopores of the supports. To confirm the adsorption of
Mucor Miehei, infrared measurements were carried out. Fig. 2 presents
the evolution of the infrared spectrum of the materials with the increase
of the concentration of Mm-L in the solution. The absorptions at
1000–1200, 800 and 450 cm−1 are assigned to the vibrations of the
silica framework. The area of the band at 800 cm−1, corresponding to
the SiOSi symmetric stretching, is used as internal standard to nor-
malize all the spectra. The major protein absorption bands due to the
peptide group's vibrations are observed in the spectral range between
1300 and 1800 cm−1. The amide I band (1600–1700 cm−1) pre-
dominantly originates from the C=O stretching vibrations of the pep-
tide bond groups and the amide II band (1510–1580 cm−1) arises from
N-H in-plane bending and C-N stretching modes of the polypeptide
chains. The amide III band arising from the N-H bending, C-Cα and C-N
stretching vibration is detected at 1460 cm−1. The intensity of the
spectral features of the lipase increase up to a lipase concentration of
10 mg/mL. We do not observe significant difference between the
spectra at 10 mg/mL and 20 mg/mL. The biocatalyst obtained by im-
mersion of SBA-15 in a 10 mg/mL kept a high specific surface (Table 1).
In the following-up of the article we will use this material as catalyst for
the transesterification reaction.

Fig. 1. SAXS patterns of SBA-15 after 210 min in the solution (pH = 6) at dif-
ferent concentrations of lipase (a) 0; (b) 5; (c) 20 mg/mL.
3.1.2. Stability
Before testing the supported biocatalyst for the transesterification
reaction, we studied the release of the enzyme. After immobilizing the
lipase by physisorption on the SBA-15 material, the material was im-
mersed in the solution at pH 6 at room temperature without stirring. A
sampling was carried out at different times and the material was ana-
lyzed by IR spectroscopy to follow the protein adsorption rate. To do
that we have considered the absorbance of the characteristic vibration

41
C. Carteret et al. Microporous and Mesoporous Materials 268 (2018) 39–45

Fig. 3. Evolution of the amount of adsorbed proteins with immersion time into
the solution at pH = 6.

band of the peptide group's in the range from 1435 to 1770 cm−1, since
no vibration in this domain is detected on the spectra of the pure silica
supports [22]. Thus, we were able to plot the evolution of the amount of
proteins adsorbed during immersion time in the solution (Fig. 3).
Under static conditions, the results depicted in Fig. 3 show a pre-
servation of the quantity of proteins adsorbed on the SBA-15 material.
There is no release phenomenon despite the weak interaction between
the lipase and the material.
3.2. Influence of reaction parameters on the transesterification process
Table 2 shows the experimental matrix for the 24 factorial design.
The last column shows the conversion of methanol to ME obtained
experimentally for each run. Four additional runs (Experiments Nos. 17
to 20) were carried out at the centerpoint to estimate the curvature
Table 2
Full Factorial design 24 with four centerpoints and responses on methyl esters
yield (methanol conversion) using immobilized lipase as catalyst.
Factor Levels used, actual (coded)
Low (−1) Medium (0) High (+1)
Fig. 4. Main effect plots: Methyl esters (ME) yield means are represented as a
function of the four factors (molar ratio, temperature, wt.% water, wt. mg
catalysts). The dotted line represents the mean for all experiments.
effect and the random error. The order in which the runs were made
was randomized to avoid systematic errors. The yields obtained at the
centerpoint show good repeatability.
Fig. 4 shows the main effects of each parameter studied. It should be
noted that the values taken by each point correspond to the average of
the yields obtained at this level, independently of the levels taken for
the other parameters. The ANOVA results allows for a statistical ana-
lysis of the contributions of the variables on the response (Table 3).
Since the p-value for the model was less than 0.05, there was a statis-
tical relation between the response and the selected variables at 95%
confidence level. Two main effects are significant (p < 0.05), with
negative effect on the (M/O) ratio and the water content (a higher yield
with a decrease in factor level). The most important parameter of the
transesterification reaction is the methanol/oil molar ratio. An increase
in this ratio results in a sharp drop in yield. Since the effect of this factor
is negative (decrease in yield when the molar ratio increases), it is

MeOH/Oil Molar Ratio, X1 1 2 3 Table 3

Temperature (°C), X2 24 34 44 Analysis of Variance (ANOVA) of the full factorial design 24.
Water content (wt. %), X3 2 5 8
Catalyst weight (mg), X4 10 15 20 Source Sum of squares dfa Mean Square F-value p-value

Model 15629.8 15 1042.0 34.76 0.002

Experiment Molar Temp. (°C) Water Catalysts Yield (%)
M/O Ratio (X1) 13908.1 1 13908.1 463.95 0.000
number Ratio wt.% wt.mg
Temperature (X2) 12.5 1 12.5 0.42 0.554
Water wt.% (X3) 253.4 1 253.4 8.45 0.044
1 1 24 2 10 75.1
Catalyst (X4) 24.2 1 24.2 0.81 0.420
2 3 24 2 20 0.4
X1.X2 48.8 1 48.8 1.63 0.271
3 1 44 2 20 71.4
X1.X3 747.1 1 747.1 24.92 0.008
4 3 44 2 10 0.5
X1.X4 22.4 1 22.4 0.75 0.436
5 1 24 8 20 47.3
X2.X3 34.0 1 34.0 1.13 0.347
6 3 24 8 10 11.1
X2.X4 20.0 1 20.0 0.67 0.460
7 1 44 8 10 45.3
X3.X4 31.2 1 31.2 1.04 0.366
8 3 44 8 20 0.7
X1.X2.X3 269.5 1 269.5 8.99 0.040
9 1 24 2 20 80.3
X1.X2.X4 21.9 1 21.9 0.73 0.441
10 3 24 2 10 0.3
X2.X3.X4 25.7 1 25.7 0.86 0.407
11 1 44 2 10 65.2
X2.X3.X4 104.7 1 104.7 3.49 0.135
12 3 44 2 20 0.3
X1.X2.X3.X4 106.5 1 106.5 3.55 0.133
13 1 24 8 10 42.6
14 3 24 8 20 11.6
Residual 119.9 4 30.0
15 1 44 8 20 70.3
Curvature 106.0 1 106.0 22.79 0.017
16 3 44 8 10 0.9
Pure error 14.0 3 4.7
17 2 34 5 15 12.8
18 2 34 5 15 12.5
Total 15749.7 19
19 2 34 5 15 13.7
20 2 34 5 15 14.9 a
Degree of freedom.

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C. Carteret et al. Microporous and Mesoporous Materials 268 (2018) 39–45

preferable to carry out the reaction using a molar ratio of 1. Then, the
water added to the reaction medium is a factor to be considered but
having a lesser effect than the molar ratio. Temperature and mass have
negligible influence on yield. A significant two-way interaction is
identified: water × (M/O). The water × (M/O) interaction reveals that
the increase of methanol conversion upon use of a lower (M/O) ratio
depends on the water concentration in the medium. For a molar ratio of
1, the increase in the percentage of water added reduces the yield. On
the contrary, for a ratio of 3, the increase of the water content leads to a
higher yield. Its (positive) effect is higher than the main effect of water
content. The highest yields are obtained when a low (M/O) ratio is
associated with low water content. Finally, a significant three-way in-
teraction is identified: (M/O) × water × Temperature. Its effect is ne-
gative. Then, the temperature should be maintained at 24 °C (low
level). In resume, the best conditions from the analysis of the experi-
mental design results are: M/O at 1, water content at 2%, and tem-
perature at 24 °C. But using these conditions the ME yield never exceeds
80% (Table 2). It should be noteworthy that a significant curvature
effect was detected (Table 3). So the ME yield does not vary linearly Fig. 6. Biodiesel synthesis: variation of the methyl esters (ME) yield as a
with one (or more) reaction parameters. In a previous work we reported function of time.
that water is essential for the activity of the immobilized lipase but an
excess of water promotes the hydrolysis reaction and induces a decrease
was undertaken. From Fig. 6, we can conclude that about 40 h are
of the transesterification yield. The ME yield varies nonlinearly with the
sufficient to achieve a yield close to 100%.
water amount [15].
We have, therefore, examined the influence of the wt.% of added
water, maintaining the most favorable conditions, that is to say, a molar 3.3.2. Gradual addition of methanol
ratio of 1, a reaction temperature of 24 °C, 10 mg of catalyst, stirring of In the optimal conditions obtained from the experimental design the
100 ppm and reaction time of 72 h. By refining the amount of water methanol conversion is close to 100% but the triglycerides conversion is
incorporated in the reaction, it was thus possible to obtain a yield close blocked to 33%. The addition of a stoichiometric amount of methanol
to 100% (Fig. 5). The optimum yield is obtained with an addition of did not make it possible to obtain a higher conversion rate of trigly-
2.5% of water in the reaction. Between 0 and 2.5%, the yield of the cerides because methanol has an inhibitory effect on lipase activity. It
reaction increases while this value of percentage of water has passed, has been demonstrated by several groups of researchers that the gra-
this yield decreases. This drop in yield is explained by the hydrolysis of dual addition of methanol makes it possible to improve the conversion
triglycerides [41,42]. To summarize, the yield of the transesterification yield of triglycerides [43–46]. Thus, a transesterification reaction was
reaction catalyzed by the physisorbed Mm-L lipase on the SBA material carried out under the optimum conditions but by adding three times
is maximal with a methanol/oil molar ratio of 1, a 2.5% water addition, one equivalent of methanol corresponding to a molar ratio of methanol
a reaction temperature of 24 °C, stirring at 100 rpm and a mass of to oil of 1. A quantity of water 2.5% was introduced at the same time as
catalyst of 10 mg. methanol. Fig. 7 shows the triglycerides conversion rate versus time.
These results indicate that it is possible to convert all the triglycerides
3.3. Optimization of operating conditions present in the oil into methyl esters by injecting three times the
equivalent of a molar ratio equal to 1, as well as 2.5% of water.
3.3.1. Kinetic study under optimized conditions However, in order to obtain such a conversion rate, nearly 200 h of
The previous experiments were carried out by fixing the reaction reaction are necessary since the methanol added must be consumed
time at 72 h. A kinetic study of the reaction under optimum conditions

Fig. 7. Triglycerides conversion rate (recovery rate) as a function of time and of

Fig. 5. Biodiesel synthesis: effect of water addition on the Methyl Ester (ME) methanol adds. Methanol add: nmethanol = ntriglycerides. All parameters are fixed
yield after 72 H. in optimal conditions: 2.5 wt% water, 24 °C, 10 mg catalyst.

43
C. Carteret et al.
Fig. 8. Recyclability of the supported catalyst under the optimized conditions.
before a new injection in order not to denature the lipase.
3.3.3. Reutilization experiment
For the evaluation of the efficiency, the production of methyl esters
of the first transesterification has been used as a reference. After each
use, the catalyst is recuperated by centrifugation, and then washed with
water in order to eliminate the glycerol. After drying, it is reused. The
recycling of the supported biocatalyst is stopped after the catalytic
activity is below 10%. From Fig. 8 it can be seen that in the optimized
conditions, even if its activity decreases rapidly, the supported bioca-
talyst can be used three consecutive runs. Infrared measurements led us
to conclude that the decrease of activity is due to a leaching of the
lipase under the operating conditions. Indeed, after the third used the
structural and textural properties of the supported biocatalyst are pre-
served but the loading of Mm-L decreases to 0.20 mg/mg, considering
the content of proteins in the commercial powder this value corre-
sponds to a quantity of adsorbed proteins equal to 4.4 10−3 mg per mg
of material. In future work, to improve the reuse of the supported
biocatalyst treatment such as silylation or crosslinking will be con-
sidered to reduce leaching.
4. Conclusion
In the present study, we have prepared supported biocatalysts by
the immobilization of Mucor Miehei lipase onto ordered mesoporous
silica materials. The hybrid materials were characterized by SAXS, ni-
trogen gas adsorption and infrared spectroscopy. Infrared spectra
proved the immobilization of the enzyme onto the silica support, and
SAXS patterns and nitrogen isotherms confirmed that the mesopore
ordering is preserved.
This supported biocatalyst is used to catalyze the transesterification
of rapeseed oil with methanol. The conditions of the transesterification
reaction of the triglycerides were optimized using the experimental
design methodology. The results indicated that methanol/oil molar
ratio, %wt water, and the interaction M/O ratio × water were the
significant factors on methyl ester yield for Mm-L-catalyzed transes-
terification of rapeseed oil. The optimum conditions of biocatalytic
transesterification reaction were temperature of 24 °C, M/O molar ratio
of 1, water amount of 2.5% wt and reaction time of 40 h. The methanol
conversion ie methyl ester yield was close to 100% under these optimal
conditions, but only 33% of the triglycerides are converted (molar ratio
of methanol to oil equal to 1). However, by (3) incremental additions of
methanol at regular intervals, nearly 100% of the oil was converted to
methyl esters in 200 h.
Acknowledgements
The work was financed by the ANR project n° ANR-09-JCJC-0024-
01.
44
Microporous and Mesoporous Materials 268 (2018) 39–45
References
[1] C. Garcia-Galan, Á. Berenguer-Murcia, R. Fernandez-Lafuente, R.C. Rodrigues,
Potential of different enzyme immobilization strategies to improve enzyme per-
formance, Adv. Synth. Catal. 353 (2011) 2885–2904.
[2] R.C. Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernandez-Lafuente,
Modifying enzyme activity and selectivity by immobilization, Chem. Soc. Rev. 42
(2013) 6290–6307.
[3] T. Jesionowski, J. Zdarta, B. Krajewska, Enzyme immobilization by adsorption: a
review, Adsorption 20 (2014) 801–821.
[4] D.N. Tran, K.J. Balkus, Perspective of recent progress in immobilization of enzymes,
ACS Catal. 1 (2011) 956–968.
[5] Y. Wan, D. Zhao, On the controllable soft-templating approach to mesoporous si-
licates, Chem. Rev. 107 (2007) 2821–2860.
[6] P. Reis, T. Witula, K. Holmberg, Mesoporous materials as host for an entrapped
enzyme, Microporous Mesoporous Mater. 110 (2008) 355–362.
[7] S. Hudson, E. Magner, J.G. Wall, Hodnett methodology for the immobilization of
enzymes onto mesoporous materials, J. Phys. Chem. B 109 (2005) 19496–19506.
[8] J. Deere, E. Magner, J.G. Wall, B.K. Hodnett, Adsorption and activity of proteins
onto mesoporous silica, Catal. Lett. 85 (2003) 19–23.
[9] Z. Zhou, M. Hartmann, Progress in enzyme immobilization in ordered mesoporous
materials and related applications, Chem. Soc. Rev. 42 (2013) 3894–3912.
[10] H. Zhang, D.R. Dunphy, X. Jiang, H. Meng, B. Sun, D. Tarn, M. Xue, X. Wang, S. Lin,
Z. Ji, R. Li, F.L. Garcia, J. Yang, M.L. Kirk, T. Xia, J.I. Zink, A. Nel, C.J. Brinker,
Processing pathway dependence of amorphous silica nanoparticle toxicity - col-
loidal versus pyrolytic, J. Am. Chem. Soc. 134 (2012) 15790–15804.
[11] S.P. Hudson, R.F. Padera, R. Langer, D.S. Kohane, The biocompatibility of meso-
porous silicates, Biomaterials 29 (2009) 4045–4055.
[12] Complementary Medicine Evaluation Committee, 16th meeting, 1999.
[13] D.Y. Zhao, J.L. Feng, Q.S. Huo, N. Melosh, G.H. Fredrickson, B.F. Chmelka,
G.D. Stucky, Triblock copolymer syntheses of mesoporous silica with periodic
50–300 angstrom pores, Science 279 (1998) 548–552.
[14] E. Magner, Immobilisation of enzymes on mesoporous silicate materials, Chem. Soc.
Rev. 42 (2013) 6213–6222.
[15] M. Kim, J. Kim, J. Lee, H. Jia, H. Na, J. Youn, J. Kwak, A. Dohnalkova, J. Grate,
P. Wang, T. Hyeon, H. Park, H. Chang, Crosslinked enzyme aggregates in hier-
archically-ordered mesoporous silica : a simple and effective method for enzyme
stabilization, Biotechnol. Bioeng. 96 (2007) 210–218.
[16] J.L. Blin, C. Gérardin, C. Carteret, L. Rodehüser, C. Selve, M.J. Stébé, Direct one-
step immobilization of glucose oxidase in well-ordered mesostructured silica using a
nonionic fluorinated surfactant, Chem. Mater. 17 (2005) 1479–1486.
[17] A. Galarneau, M. Mureseanu, S. Atger, G. Renard, F. Fajula, Immobilization of li-
pase on silicas. Relevance of textural and interfacial properties on activity and se-
lectivity, New J. Chem. 30 (2006) 562–571.
[18] A. Salis, M.F. Casula, M.S. Bhattacharyya, M. Pinna, V. Solinas, M. Monduzzi,
Physical and chemical lipase adsorption on SBA-15: effect of different interactions
on enzyme loading and catalytic performance, ChemCatChem 2 (2010) 322–329.
[19] M. Hartman, D. Jung, Biocatalysis with enzymes immobilized on mesoporous hosts:
the status quo and future trends, J. Mater. Chem. 20 (2010) 844–857.
[20] M. Mohammadi, S. Gandomkar, Z. Habibi, M. Yousefi, One pot three-component
reaction for covalent immobilization of enzymes: application of immobilized lipases
for kinetic resolution of rac-ibuprofen, RSC Adv. 6 (2016) 52838–52849.
[21] R. Sharma, Y. Chisti, U.C. Banerjee, Production, purification, characterization, and
applications of lipases, Biotechnol. Adv. 19 (2001) 627–662.
[22] J. Jacoby, A. Pasc, C. Carteret, F. Dupire, M.J. Stébé, V. Coupard, J.L. Blin, Ordered
mesoporous materials containing mucor miehei lipase as biocatalyst for transester-
ification reaction, Process Biochem. 48 (2013) 831–837.
[23] N. Canilho, J. Jacoby, A. Pasc, C. Carteret, F. Dupire, M.J. Stébé, J.L. Blin,
Isocyanate-mediated covalent immobilization of Mucor miehei lipase onto SBA-15
for transesterification reaction, Colloids Surf., B 112 (2013) 139–145.
[24] R.C. Rodrigues, R. Fernandez-Lafuente, Lipase from rhizomucor miehei as an in-
dustrial biocatalyst in chemical process, J. Mol. Catal. B Enzym. 64 (2010) 1–22.
[25] B. Norjannah, H.C. Ong, H.H. Masjuki, J.C. Juan, W.T. Chong, Enzymatic transes-
terification for biodiesel production: a comprehensive review, RSC Adv. 6 (2016)
60034–60055.
[26] L. Fjerbaek, K.V. Christensen, B. Norddahl, A review of the current state of biodiesel
production using enzymatic transesterification, Biotechnol. Bioeng. 102 (2009)
1298–1315.
[27] L.P. Christopher, H. Kumar, V.P. Zambare, Enzymatic biodiesel: challenges and
opportunities, Appl. Energy 119 (2014) 497–520.
[28] A. Gog, M. Roman, M. Tosa, C. Paizs, F.D. Irimie, Biodiesel production using en-
zymatic transesterification – current state and perspectives, Renew. Energy 39
(2012) 10–16.
[29] T. Tan, J. Lu, K. Nie, L. Deng, F. Wang, Biodiesel production with immobilized
lipase: a review, Biotechnol. Adv. 28 (2010) 628–634.
[30] M. Karimi, Immobilization of lipase onto mesoporous magnetic nanoparticles for
enzymatic synthesis of biodiesel, Biocatal. Agric. Biotechnol 8 (2016) 182–188.
[31] M. Babaki, M. Yousefi, Z. Habibi, M. Mohammadi, P. Yousefi, J. Mohammadi,
J. Brask, Enzymatic production of biodiesel using lipases immobilized on silica
nanoparticles as highly reusable biocatalysts: effect of water, t-butanol and blue
silica gel contents, Renew. Energy 91 (2016) 196 2016.
[32] Y. Yucel, Optimization of biocatalytic biodiesel production from pomace oil using
response surface methodology, Fuel Process. Technol. 99 (2012) 97–102.
[33] R.C. Rodrigues, M.A.Z. Ayub, Effects of the combined use of thermo-
myceslanuginosus and rhizomucor miehei lipases for the transesterification and
C. Carteret et al.
hydrolysis of soybean oil, Process Biochem. 46 (2011) 682–688.
[34] M.G. Jang, D.K. Kim, S.C. Park, J.S. Lee, S.W. Kim, Biodiesel production from crude
canola oil by two-step enzymatic process, Renew. Energy 42 (2012) 99–104.
[35] H. Yu, H. Yue, P. Halling, Optimal Experimental Design for an Enzymatic Biodiesel
Production System, IFAC-PapersOnLine48-8 (2015), pp. 1258–1263.
[36] C.H. Kuo, L.T. Peng, S.C. Kan, Y.C. Liu, C.J. Shieh, Lipase-immobilized biocatalytic
membranes for biodiesel production, Bioresour. Technol. 145 (2013) 229–232.
[37] H.C. Nguyen, S.H. Liang, T.T. Doan, C.H. Su, P.C. Yang, Lipase-catalyzed synthesis
of biodiesel from black soldier fly (Hermetica illucens): optimization by using re-
sponse surface methodology, Energy Convers. Manag. 145 (2017) 335–342.
[38] A. Galarneau, M. Nader, F. Guenneau, F. Di Renzo, A. Gedeon, Understanding the
stability in water of mesoporous SBA-15 and MCM-41, J. Phys. Chem. C 111 (2007)
8268.
[39] E.P. Barret, L.G. Joyner, P.P. Halenda, The determination of pore volume and area
distributions in porous substances. I. Computations from nitrogen isotherms, J. Am.
Chem. Soc. 73 (1951) 373–380.
[40] A. Galarneau, M. Mureseanu, S. Atger, G. Renard, F. Fajula, Immobilization of li-
pase on silicas. Relevance of textural and interfacial properties on activity and se-
lectivity, New J. Chem. 30 (2006) 562.
45
Microporous and Mesoporous Materials 268 (2018) 39–45
[41] L. Giraldo, J.C. Moreno-Piraján, Lipase supported on mesoporous materials as a
catalyst in the synthesis of biodiesel from Persea americana mill oil, J. Mol. Catal. B
Enzym. 77 (2012) 32–38.
[42] P. Yin, L. Chen, Z. Wang, R. Qu, X. Liu, S. Ren, Production of biodiesel by ester-
ification of oleic acid with ethanol over organophosphonic acid-functionalized si-
lica, Bioresour. Technol. 110 (2012) 258–263.
[43] M. Kaieda, T. Samukawa, T. Matsumoto, K. Ban, A. Kondo, Y. Shimada, H. Noda,
F. Nomoto, K. Ohtsuka, E. Izumoto, H. Fukuda, Biodiesel fuel production from plant
oil catalyzed by Rhizopus oryzae lipase in a water-containing system without an
organic solvent, J. Biosci. Bioeng. 88 (1999) 627–631.
[44] N. Dizge, B. Keskinler, A. Tanriseven, Biodiesel production from canola oil by using
lipase immobilized onto hydrophobic microporous styrene-divinylbenzene copo-
lymer, Biochem. Eng. J. 44 (2009) 220–225.
[45] W. Du, Y. Xu, D. Liu, J. Zeng, Comparative study on lipase-catalyzed transformation
of soybean oil for biodiesel production with different acyl acceptors, J. Mol. Catal. B
Enzym. 30 (2004) 125–129.
[46] Y. Shimada, Y. Watanabe, T. Samukawa, A. Sugihara, H. Noda, H. Fukuda,
Y. Tominaga, Conversion of vegetable oil biodiesel using immobilized Candida
Antarctica lipase, J. Am. Oil Chem. Soc. 76 (1999) 789–793.