Support information on catalysts preparation and characterization.
Full description of catalysts preparation and characterization can be find in all our published
work, in details cited in the mini-review.
In order to help the reviewers to revise the manuscript, some important information on catalysts
compared in the work are reported below.
Inorganic solid catalysts preparation and commercial information
Pure silica MCM-41 type material was prepared starting from a gel with the following
molar composition:
1 SiO2 –0.26 TMAOH - 0.12 CTABr – 40 H2O
where TMAOH is the tetramethylammonium hydroxide (mineralizing agent) and CTABr is
cetyltrimethylammonium bromide (structure directing agent).
The crystallization time and temperature were, respectively, 24 hours at 140°C in autoclave.
Delaminated zeolite ITQ-6 (Si/Al = ∞) sample was prepared by swelling the laminar pure silica
PREFER (molar gel composition: 1 SiO2 - 1.46 NH4F - 0.91 HF - 1 R - 9.1 H2O were R was the
4- amino-2,2,6,6-tetramethylpiperidine) according to the procedure described by Corma et al.
[reference 47 of the manuscript].
USY zeolite has been supplied by UOP Molecular Sieves. BEA catalyst was prepared by
hydrothermal synthesis starting from the following molar gel:
50 SiO2 – 10 TEAOH – 2 Na2O – 0.5 Al2O3 – 350 H2O
where TEAOH is tetraethylammonium hydroxide (mineralizing agent) time and temperature of
crystallization were, respectively, 5 days at 150°C in autoclave.
The molar composition of the gel for hydrothermal synthesis of FAU-X zeolites was the
following:
1 SiO2 – 0.6 Na2O – 0.1 Al2O3 – 40 H2O
After the synthesis, the solid phases of all syntheses were recovered by filtration and washed
with distilled water. The samples were calcined in air flow at 550 °C for 8 hours (heating rate: 5
°C/min). Finally, samples containing potassium (K-MCM-41, K-ITQ-6) were obtained by ionic
exchange carried out on correspondent calcined material at 60°C, for two times, with KCl 1M
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solution and a ratio solid/solution equal to 0.01 g/ml. After ionic exchange, the dried samples
were activated at 300°C for 8 hours.
Commercial potassium hydroxide (KOH) was supplied by Carlo Erba.
Commercial anhydrous potassium silicate (K2SiO3) was supplied by Alfa Aesar Co.
Commercial Amberlyst-15 (strong acidic cation exchange resin) was supplied by Sigma-Aldrich.
Enzyme-supported catalyst preparation
Enzyme used was Lipase from Rhizomucor Miehei (RML), commercial Palatase 20000L, SigmaAldrich. The enzyme structure is reported below:
Enzyme-adsorption and covalent attachment procedures
In 50 ml of a 0.2 M phosphate buffer solution (pH 7), the lipase and the calcined support
(Silicalite-1 and ITQ-2 for adsorption, functionalized Sepiolite AlPO4 materials for covalent
attachment) were mixed and stirred at 250 rpm for 24 h at 0°C. The support with immobilized
lipase was separated by filtration, washed twice with de-ionized water and dried at 25°C
overnight. The total protein concentration of the initial solution and the supernatant was
calculated using the UV Absorption Methods (at 280 nm).
Enzyme-encapsulation procedure
The enzyme encapsulation procedure was as follows: a determined amount of lipase solution was
added to the CTABr (cetyltrimethylammonium bromide) solution and stirred for 1 hour at room
temperature. The silica precursor was then introduced into the solution and, subsequently,
ethanolamine (20 wt%) was added. The gelation was slow and the sol-gel was stirred for 24
hours at room temperature and pH = 7.2. The final molar gel composition was: 1 SiO2 – 0.16
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CTABr – 0.05 Ethanolamine – 0.020 Enzyme – 40 H2O. The mixture was filtered and the liquids
were analyzed by UV-Adsorption at 280 nm to determine the degree of enzyme encapsulation.
The powder containing the enzyme was stored at 4°C until use. A scheme of encapsulated
enzyme is shown below:
Liposome hybrid-nanospheres containing enzyme preparation procedure
The synthesis of hybrid nanospheres consists in two operative and subsequently stages:
preparation of liposome nanospheres encapsulating enzyme and formation of an inorganic
porous silica shell around the organic nanospheres. A scheme of the preparation route is shown
below:
A specific amount of lecithin was mixed with chloroform in order to dissolve it and to create a
homogeneous solution. The solution was vigorously stirred for about 10 minutes, until to obtain
an emulsion of yellow color. The solution was then placed in a rotavapor to remove the
chloroform. Thereby, this solution was mixed with 40 mL of 0.2 M phosphate buffer pH=7
containing 7.1 mL of lipase. The suspension was maintained during 2 hours at 40°C with
continuous stirring. Finally, in order to obtain homogeneous nanospheres, the liposomal/enzyme
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solution was extruded two times using cellulose filters with a pores diameter equal to 0.20
micron. During the second stage, an external inorganic shell (silica porous matrix) was formed
around the liposomes/enzyme nanospheres, previously synthesized: 0.44 g of tetraethyl
ortosilicate were added to the 47 ml of liposomal/enzyme solution. The suspension formed was
stirred at 350 rpm, at room temperature, for 2 hours and following, 7.1 mg of NaF was
incorporated to initializing the condensation of silane groups. The stirring was maintained for 48
hours at room temperature. To separate the supernatant from the medium containing the enzyme
(hybrid nanospheres), the mixture was centrifuged at 15000 rpm for 30 minute. The total protein
concentration (C, [mg/mL]) of the initial solution and of the supernatant was calculated using
UV absorption method at 280/205 nm. The recovered solid was washed with distilled water,
dried at 30°C overnight and stored at 0°C to preserve the functionality of the enzyme until its
catalytic use.
Catalysts characterization
Powder X-ray diffraction (XRD) data were recorded using a Phillips PW 1710
diffractometer with CuK radiation. The samples were scanned in the range of 2 from 1 to 8°
(for mesoporous materials) or from 5 to 45° (for microporous materials) in steps of 0.005° with a
count time of 1 s at each point. XRD analyses of all synthesized samples show the characteristic
diffraction peaks (patterns not reported), confirming that the expected phases have been obtained
for all materials. BET surface area and physical properties of samples were evaluated by N 2
adsorption/desorption isotherms carried out at 77 K on a Micromeritics ASAP 2020 sorption
analyser. Thermal decompositions of as-synthesized samples were investigated by SHIMADZU
DTG-60 instrument, between 20-850 °C, at a ramp of 5 °C/min in air with a flow rate of 5
ml/min. Chemical composition of samples was evaluated by an Elan DRC-e ICP-MS instrument
(Perkin-Elmer SCIEX). Samples were introduced by means of a quartz nebulizer. The ICP torch
was a standard torch (Fassel type torch) with a platinum injector. For the quantitative analysis,
calibration curves were build on six different concentrations in a calibration range of 1-5000 μg/l
and having composition similar to that of solution samples. Standard solutions were prepared by
diluting a solution of Na and K, (1000 mg/l). For IR measurements, self-supporting wafers were
prepared and activated under dynamic vacuum (10-4 Torr) for one hour at 573 K, in an IR cell
allowing in-situ thermal treatments, gas dosage and IR measurements to be carried out both at
room temperature and at a nominal temperature of 77 K, presumably in fact around 100 K.
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Spectra were collected on a Bruker IFS 55 Equinox instrument equipped with a MCT
cryodetector working with 2 cm-1 resolution. Difference spectra are obtained after subtraction of
the spectrum of the naked sample.
Transesterification reaction procedure
The alcoholysis of triglycerides with anhydrous methanol (99,9%, Sigma-Aldrich) catalyzed by
acid or base catalysts was performed in batch teflon-steel autoclaves immersed in a temperaturecontrolled bath. The reaction temperature was varied from 70°C to 180°C.
The catalytic tests catalyzed by enzyme were carried out stirring the reaction system at 350 rpm
and at 37 °C (optimal reaction temperature for lipase activity), for all reaction time.
To analyze the reaction progress, products and reactant were separated from catalyst and
glycerin by centrifugation in hexane (95%, Sigma-Aldrich). To analyze the chemical
composition of the biodiesel produced we used the ASTM D-6584-00 method. Through this
method, it is possible to quantify triglycerides (TG), diglycerides (DG), monoglycerides (MG),
free fatty acids (FFA) and methyl ester by unique chromatogram. The Rtx®-Biodiesel fused
silica capillary column (10 m x 0.32 mm x 0.10 µm) was used in an Agilent 6890 GC instrument
equipped with FID detector. Before analysis, trimethylsilyl-trifluoroacetamide (98%+ Acros
Organics) was added to the mixture as silyating agent. Tricaprin (Sigma-Aldrich, puriss. p.a.
standard for GC) was added as external standard. 1µL of sample was injected and analyzed by
the following oven temperature program: 1 minute at 140°C, 10°C/min to 360, 8 minutes at
360°C. To correct the area obtained from the GC, in order to calculate the exact triglycerides
conversion and biodiesel yield, the response factors for each compounds have been calculated
using the correspondent standard compound (puriss. p.a. standard for GC): oleic acid, methyl
oleate, monoglycerides, diglycerides and triolein. The biodiesel yield and the triglycerides
conversion were calculated according to the following equations:
where the A indicated the peak area of the component, corrected by corresponding response
factor, while the subscript indicates the particular component: AMM = Correct Area of methyl
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myristate, AMO = Correct Area of methyl oleate, AMP = Correct Area of methyl palmitate, AMA =
Correct Area of myristic acid, APA = Correct Area of palmitic acid, AOA = Correct Area of oleic
acid, AMG = Correct Area of monoglycerides, ADG = Correct Area of diglycerides and ATG =
Correct Area of triglycerides.
After the reaction time, two centrifugation processes were carried out in order to separated the
catalyst from the reaction mixture and the glycerol to biodiesel mixture. All recovered catalysts
were washed two times with n-hexane and distillated water, and after dried at 30°C (for
biocatalyst) or at 120°C (for inorganic catalyst) overnight, the catalyst was ready for next
catalytic reaction cycle.
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