Efficient bio-conversion of glycerol to glycerol carbonate catalyzed by lipase extracted from Aspergillus niger

Article abstract of DOI:10.1039/c2gc16294f

A biocatalytic synthesis of glycerol carbonate (GlyC), as an added-value product of renewable glycerol, has been developed using a catalytic route in which glycerol (Gly) was reacting with dimethyl carbonate (DMC) in the presence of lipase under solvent-free conditions. The enzyme screening indicated lipase from Aspergillus niger as the most efficient biocatalyst for the GlyC synthesis. After the optimization of the reaction conditions it was established that the best results corresponded to 12% (w/w) Aspergillus niger lipase, to a glycerol:DMC molar ratio of 1:10, to an incubation time of 4 h and to an incubation temperature of 60 °C. Consequently, the glycerol conversion was around 74%, the yield in GlyC of 59.3% and the selectivity to GlyC of 80.3%. Recycling experiments demonstrated that the biocatalyst can be successfully used for several reaction cycles (at least 4 times) and confirmed its very high stability under the reaction conditions.

Full text of DOI:10.1039/c2gc16294f

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Green Chemistry
Cite this: Green Chem., 2012, 14, 478
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PAPER
Efficient bio-conversion of glycerol to glycerol carbonate catalyzed by lipase
extracted from Aspergillus niger
Madalina Tudorache, Loredana Protesescu, Simona Coman and Vasile I. Parvulescu*
Received 17th October 2011, Accepted 21st November 2011
DOI: 10.1039/c2gc16294f
A biocatalytic synthesis of glycerol carbonate (GlyC), as an added-value product of renewable
glycerol, has been developed using a catalytic route in which glycerol (Gly) was reacting with
dimethyl carbonate (DMC) in the presence of lipase under solvent-free conditions. The enzyme
screening indicated lipase from Aspergillus niger as the most efficient biocatalyst for the GlyC
synthesis. After the optimization of the reaction conditions it was established that the best results
corresponded to 12% (w/w) Aspergillus niger lipase, to a glycerol : DMC molar ratio of 1 : 10, to
◦
an incubation time of 4 h and to an incubation temperature of 60 C. Consequently, the glycerol
conversion was around 74%, the yield in GlyC of 59.3% and the selectivity to GlyC of 80.3%.
Recycling experiments demonstrated that the biocatalyst can be successfully used for several
reaction cycles (at least 4 times) and confirmed its very high stability under the reaction conditions.
Introduction
GlyC represents the main electrolyte ingredient of lithium-
based batteries and can be used for the production of coatings,
6
The current requests of the chemical industry assume the
development of green synthetic routes involving renewable raw
materials and the replacement of “unfriendly” syntheses, which
usually generate toxic residues and/or involve harsh reaction
conditions, with friendly ones. Nowadays glycerol is generated as
a by-product at low prices (e.g. from the production of biodiesel
using biomass as a raw material). However, it is considered as one
of the main candidates to be used as a building block in chemical
synthesis. Thus, it is already considered as a platform molecule
for fine syntheses, and the possibility to transform it into a
large number of high-value chemicals, such as classic esters and
oligomers, and also into new products like telomers, branched
alkyl ethers, propanediols, epoxides and glycerol carbonate has
adhesives and lubricants via polymerization or via reaction with
izocyanates/acrylates.
7
Nowadays, the industrial production of GlyC takes place
following two reaction steps involving the synthesis of a cyclic
1
ethylene carbonate followed by its reaction with glycerol.
However, these procedures present several economic and
environmental drawbacks that require improvements to achieve
the desired feasibility. A possible alternative for the green
production of GlyC is glycerolation of dimethyl carbonate
(DMC) with glycerol (Gly) using either inorganic or bio-
catalysts (Scheme 1). Glycerol has two types of OH groups
exhibiting different reactivity: the two primary alcohol groups
are presumably more reactive than the secondary OH group.
Accordingly, the reaction mechanism of glycerolation is sup-
posed to follow the route described in Scheme 1. Glycerol
carboxylation leads to an unstable intermediate (3), which forms
the GlyC (4) product through an intramolecular conversion. The
reaction can continue to glycerol dicarbonate (GlyDC) (5) and
furthermore to diglycerol tricarbonate (DGlyTC) (6) when the
1,2
been proved.
Glycerol carbonate (4-hydroxymethyl-1,3-dioxolan-2-one,
GlyC) is a relatively new compound in the chemical industry
with great potential in a large synthesis area. Thus GlyC is
considered a new “green” solvent due to its ideal physico-
chemical properties, such as high stability, low toxicity, low
3
evaporation rate and low flammability and the possibility to use
7
excess of DMC is substantially high.
it as a solvent in cosmetics, personal care items, and medicine
Both reagents (e.g. Gly and DMC) are non-toxics, biodegrad-
ables and are generated through clean production processes.
Chemo-catalytic synthesis of GlyC based on the reaction of Gly
with DMC has been investigated in both homogeneous con-
4
has been suggested. GlyC is also a valuable intermediate for
the production of resins and plastics, and in the pharmaceutical
5
and cosmetics industry after its transformation into glycidol.
7
ditions using alkali-based catalysts (e.g. potassium carbonate)
and heterogeneous conditions using solid calcium materials or
University of Bucharest, Department of Organic Chemistry, Biochemistry
and Catalysis, Bd. Regina Elisabeta 4–12, Bucharest, 030016, Romania.
E-mail: vasile.parvulescu@g.unibuc.ro; Fax: 4021 4100241; Tel: 4021
precursors of these (e.g. calcium oxide, calcium hydroxide or
8,9
calcium methoxide). Recently, for the synthesis of GlyC, the
4
100241
use of lipases (extracted from Candida antarctica) was reported
4
78 | Green Chem., 2012, 14, 478–482
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antarctica. After the selection of the most performant enzyme
Aspergillus niger lipase), the second objective was to optimize
(
the synthesis conditions and to check the recyclability, and
implicitly, the stability of the bio-catalyst.
Results and discussion
Screening of biocatalyst for glycerol carbonate synthesis
The catalytic activity of lipase from various natural sources
has been directly evaluated in the transesterification of glycerol
with DMC under solvent-free conditions. Using this strategy,
the influence of DMC, as solvent, on the lipase activity has
also been evaluated. The collected data showed that among
the tested lipases, Pseudomonas cepacia, Candida antarctica and
Aspergillus niger were the only sources expressing lipase enzyme
able to conserve at a high level the catalytic activity in DMC
solvent (Fig. 1). These data confirm results already reported
showing Candida antarctica as an efficient lipase source for the
10–12
GlyC synthesis.
As it was mentioned above this was used as
reference in our study. However, the screening of the lipases
presented in Fig. 1 showed that Aspergillus niger represents
a more efficient lipase source then Pseudomonas cepacia and
Candida antarctica. Therefore, Aspergillus niger was selected for
the optimization of the GlyC synthesis. All other sources led to
poorer results (Fig. 1).
Fig. 1 Screening the lipase sources for glycerol carbonate synthesis.
Reaction conditions: 0.1 g glycerol, 1 mL DMC and 0.02 g enzyme;
◦
temperature 60 C; 48 h incubation time.
Optimization of the reaction conditions
Scheme 1 Glycerol carbonate (GlyC) synthesis based on DMC glyc-
erolation with glycerol (Gly).
Fig. 2 shows results for the synthesis of GlyC using different
molar ratios of Gly to DMC. Theoretically, the reaction
stoichiometry requires an equimolar ratio of reagents (i.e. of
10,11
as well,
thus moving the reaction conditions to the mild ones
and the selectivity to high values. One of these reports proposed
the transesterification of glycerol with DMC in tetrahydrofuran
11
(
THF). The second one indicated the synthesis of GlyC
under solvent-free conditions with DMC playing a double role:
10
substrate and solvent.
In this study, we extended the scope of the synthesis of
GlyC under solvent-free conditions. We report a screening of
the catalytic performances of various lipases extracted from
Aspergillus niger, Pseudomonas fluorescens, Phizopus arrhizus,
Candida cylindracea, Pseudomonas cepacia, Mucor miehei, As-
pergillus sp, Porcina pancreas, Phizopus niveus, Hog pancreas and
Thermomyces lonuginosus. The experiments have been carried
out considering as reference a lipase extracted from Candida
Fig. 2 Influence of reagents molar ratio on the synthesis of GlyC (ꢀ
– glycerol conversion (%), ꢁ – GlyC selectivity (%)). Conditions: 0.1 g
◦
glycerol; 12% (w/w) lipase; 60 C temperature; 48 h incubation time.
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Gly-to-DMC, see Scheme 1). However, the mechanism of the
reaction is strongly influenced by the catalyst type (e.g. inorganic
structure or enzyme) and the reaction conditions. Base inorganic
selectivity and yield). It was thus found that a high enzyme
density causes inter-molecular interactions of lipase, in this way
blocking the catalytic sites. Comparing the data with Aspergillus
niger lipase obtained in this study with those already reported
in the literature with the lipase from Candida Antarctica (either
catalysts (e.g. K
2
CO ) favor the formation of GlyC using only
3
a 3 fold excess of DMC. Secondary products (e.g. GlyDC and
11
DGlyTC) resulted for a higher excess of DMC (10 fold) and at
in THF (21% (w/w) or under free-solvent conditions (33%
◦
7
10
high temperatures (e.g. 95 C). Using enzymes, the optimum
molar ratio of Gly to DMC that has been reported was 1 : 2
when the reaction was carried out in THF solvent, and a much
(w/w) ) it appears that the lipase from Aspergillus niger is,
indeed, a more efficient catalyst.
10,11
smaller (1 : 10) for solvent-free systems.
Effect of incubation time and temperature on GlyC synthesis
The synthesis of GlyC in the presence of Aspergillus niger
lipase followed the same tendency. Thus, for an equimolar
ratio of Gly to DMC, the Gly conversion was of only 46.4%
without the formation of the GlyC product (Fig. 2). In this
case, we suppose that only one glycerol “–OH” group reacts
with DMC. Consequently, the DMC could couple two glycerol
molecules due to the glycerol excess. This supposition is in
agreement with the mechanism of DMC glycerolation proposed
Fig. 4 shows the variation of the glycerol conversion, yield in
GlyC and selectivity to GlyC after different incubation times.
The reaction almost establishes the equilibrium after 4 h (74%
Gly conversion, 58% GlyC yield and 80% GlyC selectivity).
Further increase of the incubation time to 48 h led to an
increase of the conversion with 10%, while the yield remained
nearly the same (Fig. 4). However, the selectivity is a key
factor of the process and the increase of the incubation time
to 48 h generated a continuous decrease due to the formation
of secondary products. It appears thus that an incubation time
of 4 h provides the best compromise between the conversion of
Gly and the selectivity to GlyC. It is also worth to mention that
using Candida antarctica lipase a conversion of glycerol of 74%
7
by Rokicki et al. The decrease of the Gly : DMC ratio led to
both an increase of the conversion and to the formation of
the desired product with an increased selectivity. Indeed, it was
found that a molar ratio of 1 : 10 corresponds to an optimum
under these conditions. For Aspergillus niger it corresponded to
a Gly conversion of 73.8% and a selectivity to GlyC of 80%.
Further decrease of this ratio till a Gly : DMC of 1 : 100 led to
only a very small increase of the Gly conversion (80%) but to a
significant depreciation of the GlyC selectivity (62%).
10
was achieved only after 25 h.
Fig. 3 illustrates the effect of the enzyme concentration in
this reaction. The plot profile indicates a gradual increase of
the process efficiency (Gly conversion and yield in GlyC) when
the enzyme concentration raise from 5 to 12% (w/w) (Gly
conversion of 81.3% and yield in GlyC of 60.4% for 12% lipase
concentration (w/w)). The selectivity suffers a slight decrease in
this range of catalyst concentrations (around 25%). A further
increase of the enzyme loading till 15% (w/w) had no effect on
Gly conversion and selectivity to GlyC, but the yield to GlyC
decreased with around 5%. 12% (w/w) seems to be not only
an economic limit of the catalyst loading, but also in terms
of performances. Thus, higher concentrations than 15% (w/w)
led to a decrease of the monitored parameters (conversion,
Fig. 4 Influence of the incubation time on the GlyC synthesis (ꢀ –
glycerol conversion (%), – GlyC yield (%), ꢁ – GlyC selectivity (%)).
Conditions: 0.1 g glycerol; glycerol : DMC molar ratio = 1 : 10; 12%
◦
(
w/w) lipase; temperature – 60 C.
The influence of the temperature on the efficiency of the GlyC
synthesis has also been investigated. The experimental results are
summarized in Fig. 5. Both the conversion of Gly and the yield to
GlyC increased gradually with the temperature till a maximum
◦
at 60 C (73.8% conversion of Gly and 59.3% yield to GlyC).
◦
Higher temperatures than 60 C led to a drastic decrease of
the reaction performances, most likely, due to the denaturation
13
of the enzyme structure. Also, the selectivity to GlyC showed
a slow decrease when the temperature varied between 25 and
◦
6
0 C (from 95% to 80%, respectively). However, the selectivity
of the process presented a sudden drop for temperatures higher
◦
than 60 C, which also corresponds to an enzyme inhibition by
structure denaturation.
Fig. 3 Lipase concentration in the reaction mixture (᭜ – glycerol con-
The TOF (turnover frequency) of the lipase from Aspergillus
niger under optimum conditions of the GlyC synthesis was of
1.16 ¥ 10 h .
version (%), ᭹ – GlyC yield (%), ꢀ – GlyC selectivity (%)). Conditions:
◦
0
4
.1 g glycerol; glycerol : DMC molar ratio = 1 : 10; temperature 60 C;
5
-1
8 h incubation time.
4
80 | Green Chem., 2012, 14, 478–482
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◦
under mild conditions (60 C and atmospheric pressure), with
a convenient atomic economy; (3) it leads to a performant
synthesis process for GlyC production (conversion of glycerol
of 73.8%, yield to GlyC of 59.3% and selectivity to GlyC of
8
0.3%).
Experimental
Chemicals and enzymes
The substances used in this study: glycerol (Gly) and dimethyl
carbonate (DMC) were purchased from Sigma-Aldrich (USA).
Lipase enzymes from different sources (e.g. Aspergillus niger,
Candida antarctica, Pseudomonas fluorescens, Phizopus arrhizus,
Candida cylindracea, Pseudomonas cepacia, Mucor miehei, As-
pergillus sp, Porcina pancreas, Phizopus niveus, Hog pancreas
and Thermomyces lonuginosus) were also obtained from Sigma-
Aldrich (USA). Derivatization reagents (BSTFA : TMCS =
Fig. 5 Temperature effect on the evolution of the GlyC synthesis
process ( – glycerol conversion, ꢁ – yield in GlyC, ꢀ – selectivity
to GlyC). Conditions: 0.1 g glycerol; glycerol : DMC molar ratio = 1 : 10;
◦
1
2% (w/w) lipase; temperature 60 C; 48 h incubation time.
Biocatalyst stability and recyclability
99 : 1 and pyridine) were purchased from Macherey-Nagel
study involves a two phase configuration with DMC
Corp. (Duren, Germany) and Fluka (Switzerland). The or-
ganic solvents used in all the experiments were of analytical
purity.
The synthesis of GlyC using the methodology reported in
this study involves a two phase configuration with DMC corre-
sponding to the hydrophobic phase and Gly to the hydrophilic
phase. The biocatalyst (Aspergillus niger lipase) was dispersed in
the reaction volume. At the end of the reaction, the enzyme was
easily recovered from the reaction mixture by centrifugation and
the catalyst was recycled in another GlyC synthesis. The opera-
tional stability of Aspergillus niger lipase was thus investigated in
five successive reaction cycles (Table 1). The experimental data
showed no significant changes in the biocatalyst activity after
four cycles. Till the fourth cycle, the loss of the conversion was
of maximum 4% while the yield in GlyC decreased with less
than 5%. However, after the fourth cycle the enzyme rapidly
lost the activity. Both the Gly conversion and the yield in GlyC
were diminished under these conditions to half of the initial
values (Table 1). The behavior is reproducible and requires more
characterization evidence to explain the abrupt behavior.
Bio-catalytic synthesis of glycerol carbonate (GlyC)
Enzymatic synthesis of GlyC has been performed in a solvent-
free system. Given amounts of Gly and DMC at different
Gly : DMC molar ratios (1 : 5, 1 : 10, 1 : 25, 1 : 50 and 1 : 100)
were mixed together with the lipase catalyst (2.58, 5.04, 7.38,
11.72, 15.15, 21.13, 26.32%, w/w) in a 1.5 mL reaction vial
(
Eppendorf tube). The mixtures were incubated for a maximum
◦
of 48 h under stirring at temperatures in the range 30–80 C using
a thermostatted shaker. After the reaction was completed, the
suspension was centrifuged to recover the enzyme. The filtrated
◦
liquid phase was evaporated at 50 C under vacuum for the
elimination of the DMC excess and methanol. Finally, only
dried reaction products (e.g. GlyC and secondary products) and
unreacted Gly were found in the vial. The recovered enzyme was
used for the next round of the reaction.
Conclusions
A solvent-free synthesis of GlyC has been developed based on
glycerolation of DMC catalyzed by a lipase enzyme from an
Aspergillus niger source. The main advantages of the developed
synthesis method are (1) the strategy follows an eco-friendly
and non-toxic route (i.e. the reaction mixture before and after
synthesis does not contain pollutants); (2) it corresponds to
an efficient synthesis since a low enzyme concentration is
required (only 12% (w/w) lipase) and the enzyme stability
and recyclability under operation conditions have been proved
for four reaction cycles; the chemical reaction was carried out
Analyses
The reaction products were analyzed using gas chromatography
(
(
GC) coupled to mass-spectrometry (MS) or flame ionization
FID) detectors. While GC-MS allowed the identification of
the reaction products, GC-FID led to a quantitative evalu-
ation of the reaction mixture composition at the end of the
synthesis.
The analysis of glycerol and reaction products required
silylation before the injection on the chromatographic col-
umn, in order to obtain proper peak shapes, and also a low
14
detection limit. For this purpose, 100 mL silylation agent
(BSTFA : TMCS = 99 : 1) were added to the reaction samples
Table 1 The stability and recyclability of Aspergillus niger lipase in
GlyC synthesis. Conditions: 0.1 g glycerol; glycerol : DMC molar ratio =
◦
1
: 10; 12% (w/w) lipase; temperature 60 C; 1 h incubation time
(
after the evaporation step), and then the resulted mixture was
diluted with 100 mL pyridine. The derivatization process has
been performed under gently agitation, at 60 C, for 30 min.
Before analysis, 100 mL of n-heptane was added as internal
standard.
Batch no.
1
2
3
4
5
◦
a
C
Gly (%)
62.6
52.6
58.9
53.5
58.3
49.7
58.1
47.2
27.7
27.0
GlyC yield (%)
a
Derivatized samples (1 mL) were analyzed with a GC-FID
Schimadzu GC-2014, Thermo Electron Scientific Corporation,
C
Gly – glycerol conversion.
(
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USA) chromatograph equipped with TR-WAX and TR1MS
spectra of the samples containing GlyC exhibited a band at
-
1
-1
capillary columns using hydrogen as a carrier gas (1.0 mL min ).
The detector and injector were set up at the temperature of
1789 cm corresponding to carbonyl stretching mode of glycerol
9,15
carbonate.
◦
◦
2
50 C. The temperature in the oven was kept constant at 50 C
◦
◦
-1
for 1 min and then was increased to 250 C with a 10 C min
rate. Finally, the oven temperature was maintained at 250 C for
Acknowledgements
◦
This work was financially supported by PN II HR YT-96
program, contract no. 91/2010 from CNCSIS.
3
min. GC-MS analyses were performed with a Trace GC 2000
system with MS detector (Thermo Electron Scientific Corpo-
ration, USA) incorporating a TR-WAX capillary column. The
injection chamber was set up at 200 C and the temperature in
◦
References
◦
the detector cell was 270 C. The oven program was similar with
1
A. Behr, J. Eilting, K. Irawadi, J. Leschinski and F. Lindner, Green
Chem., 2008, 10, 13–30.
that used for the GC-FID analysis. The GC-MS chromatogram
and the corresponding spectra of silylated samples were used to
identify the reaction products. MS (m/z) (1) silylated glycerol
carbonate: 190 (M ), 145 (M - 3CH
1
2 M. Pagliaro, R. Ciriminna, H. Kimura, M. Rossi and C. Della Pina,
Angew. Chem., Int. Ed., 2007, 46, 4434–4440.
3 J. Huntsman Corporation, Glycerine Carbonate, 2009, http://
www.huntsman.com/performanceproducts/Media/JEFFSOL%
C2%AEGlycerineCarbonate.pdf.
+
+
+
3
), 131 (M - Si(CH
3
)
2
+
),
),
+
+
+
17 (M - Si(CH
3
)
3
), 101 (M - OSi(CH
3
)
3
), 89 (OSi(CH
)
3 3
+
9
7
3 (Si(CH
3
)
3
).
4 D. Herault, A. Eggers, A. Strube and J. Reinhard, Ger. Offen.,
2
002, DE 10110855.
The conversion of Gly (CGly), the yield in GlyC and the
5
J. Rousseau, C. Rousseau, B. Lynikaite, A. Sakcus, C. de Leon, P.
selectivity to GlyC were calculated using equations (1–3), where
the number of moles was determined from the chromatographic
analysis.
Rollin and A. Tatibouet, Tetrahedron, 2009, 65, 8571–8581.
6 A. S. Kovvali and K. K. Sirkar, Ind. Eng. Chem. Res., 2002, 41,
2
287–2295.
7
G. Rokicki, P. Rakoczy, P. Parzuchowski and M. Sobiecki, Green
C
Gly(%) = (sum of the moles of reaction products)/(moles
of Gly introduced in the reaction) ¥ 100
Chem., 2005, 7, 529–539.
(1)
(2)
(3)
8 J. Li and T. Wang, React. Kinet., Mech. Catal., 2011, 102, 113–126.
9
J. R. Ochoa-G o´ mez, C. Ram ´ı rez-L o´ pez, M. C. Villar a´ n-Velasco,
L. Lorenzo-Ibarreta, A. Pesquera-Rodr ´ı guez, O. G o´ mez-Jim e´ nez-
Aberasturi, J. Torrecilla-Soria and B. Maestro-Madurga, Appl.
Catal., A, 2009, 366, 315–324.
GlyC yield(%) = (moles of GlyC/moles of Gly introduced
in the reaction) ¥ 100
1
0 E. Y. Lee, K. H. Lee and C.-H. Park, Bioprocess Biosyst. Eng., 2010,
3, 1059–1065.
3
GlyC selectivity (%) = (moles of GlyC/sum of the moles of
reaction products) ¥ 100
11 D. Y. Yoon, S. C. Kim, H. Lee, B. K. Song and Y. H. Kim, J. Mol.
Catal. B: Enzym., 2007, 49, 75–78.
1
2 D Royon, M Daz, G Ellenrieder and S. Locatelli, Bioresour. Technol.,
2007, 98, 648–653.
The characterization of the reaction products was also
performed by FTIR spectrometry using a Thermo 4700 spec-
trometer (Thermo Electron Scientific Corporation, USA). The
samples were analyzed after the DMC excess has been re-
moved (dried samples prepared under vacuum conditions). The
13 R. Sharmaa, Y. Chistib and U. Chand Banerjee, Biotechnol. Adv.,
001, 19, 627–662.
2
1
1
4 C. Plank and E. Lorbeer, J. Chromatogr., A, 1995, 697, 461–468.
5 M. Aresta, A. Dibenedetto, F. Nocito and C. Ferragina, J. Catal.,
2009, 268, 106–114.
4
82 | Green Chem., 2012, 14, 478–482
This journal is © The Royal Society of Chemistry 2012