A cyclic process for full enzymatic saccharification of pretreated cellulose with full recovery and reuse of the ionic liquid 1-butyl-3- methylimidazolium chloride

Article abstract of DOI:10.1039/c2gc35905g

A sustainable cyclic process for the enzymatic saccharification of ionic liquid (IL)-pretreated cellulose, in which the IL is recovered and recycled, has been developed. Homogeneous cellulose solutions in the IL 1-butyl-3- methylimidazolium chloride ([Bmim][Cl]) were used to prepare amorphous cellulose by antisolvent precipitation with water, ethanol or equimolar water-ethanol mixtures as green molecular solvents. Several operation parameters (e.g., solvent, temperature, ultrasounds, etc.) for both cellulose precipitation and the washing steps were tested to achieve full desorption of the IL from the cellulose backbone. In the best conditions, up to 99.7% IL was recovered, which was then successfully reused in further cellulose dissolution/precipitation cyclic processes. Furthermore, the cellulose regenerated in each cycle was an excellent substrate for enzymatic hydrolysis, permitting full hydrolysis (i.e., up to 97.7% hydrolysis after 4 h at 50 °C) by the combined action of both cellulase and cellobiase enzymes, that provides a clear glucose solution. The excellent suitability of this glucose solution for growing aerobic Saccharomyces cerevisiae was demonstrated.

Full text of DOI:10.1039/c2gc35905g

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PAPER
A cyclic process for full enzymatic saccharification of pretreated cellulose with
full recovery and reuse of the ionic liquid 1-butyl-3-methylimidazolium
chloride†
a
a
a
b
Pedro Lozano,* Berenice Bernal, Imanol Recio and Marie-Pierre Belleville
Received 14th June 2012, Accepted 10th July 2012
DOI: 10.1039/c2gc35905g
A sustainable cyclic process for the enzymatic saccharification of ionic liquid (IL)-pretreated cellulose, in
which the IL is recovered and recycled, has been developed. Homogeneous cellulose solutions in the IL
1
-butyl-3-methylimidazolium chloride ([Bmim][Cl]) were used to prepare amorphous cellulose by
antisolvent precipitation with water, ethanol or equimolar water–ethanol mixtures as green molecular
solvents. Several operation parameters (e.g., solvent, temperature, ultrasounds, etc.) for both cellulose
precipitation and the washing steps were tested to achieve full desorption of the IL from the cellulose
backbone. In the best conditions, up to 99.7% IL was recovered, which was then successfully reused in
further cellulose dissolution/precipitation cyclic processes. Furthermore, the cellulose regenerated in each
cycle was an excellent substrate for enzymatic hydrolysis, permitting full hydrolysis (i.e., up to 97.7%
hydrolysis after 4 h at 50 °C) by the combined action of both cellulase and cellobiase enzymes, that
provides a clear glucose solution. The excellent suitability of this glucose solution for growing aerobic
Saccharomyces cerevisiae was demonstrated.
methods have been employed to improve the accessibility of
biomass polysaccharides to enzymatic hydrolysis (e.g., ball and
compression milling, dilute acids and hydrothermal treatment,
bases, etc.), however, none are able to increase the surface
area and decrystallize cellulose sufficiently to permit full
enzymatic hydrolysis, short residence times and low enzyme
1
Introduction
The production of second generation bioethanol from non-edible
biomass (e.g., lignocellulosic biomass) using clean and sustain-
able approaches is one of the greatest challenges on the research
1
and industrial agenda. Bioethanol production from cellulosic
3
sources consists of three consecutive steps, such as, the pretreat-
ment of cellulose to disrupt its highly ordered and rigid structure,
hydrolysis of the cellulose to fermentable sugars, and finally, the
ethanol fermentation by microorganisms. Full enzymatic
hydrolysis of cellulose to its glucose units can be carried out by
the synergistic action of different glycohydrolases, such as cellu-
lases (endoglucanases EC 3.2.1.4, exoglucanases EC 3.2.1.91)
and cellobiase (EC 3.2.1.21). The high specificity of enzymes is
the characteristic that identifies them as the greenest approach for
concentrations.
The pioneering work of Rogers’ group, showing that some
ILs, such as 1-butyl-3-methylimidazolium chloride ([Bmim]-
[
Cl]), 1-hexyl-3-methylimidazoium chloride ([Hmim][Cl]), etc.,
4
are able to dissolve cellulose, has opened up new opportunities
for the valorisation of large amounts of waste cellulose-contain-
ing materials (e.g., forest biomass). Among those the biocatalytic
depolymerisation of cellulose to its glucose units, and their sub-
sequent transformation into bioethanol by fermentation, is the
2
5
the saccharification of cellulose. However, the crystalline struc-
most widely attempted.
ture of cellulose, which is supported by multivalent inter- and
intramolecular hydrogen bonds, involves a recalcitrance to its
degradation by biocatalysts. Several chemical and physical
Although most ILs have been shown to act as excellent reac-
tion media for enzyme-catalyzed reactions, it has been widely
reported that ILs that are excellent for dissolving cellulose (e.g.,
6
[
Bmim][Cl], etc.), produce fast enzyme deactivation by protein
7
unfolding. In this context, alternative approaches to overcome
the negative effect of ILs-dissolving cellulose on cellulase
activity and stability have been assayed for cellulose saccharifi-
cation, e.g., by using buffered media containing “benign” dis-
solved ILs at low concentrations, such as dialkylphosphate or
a
Departamento de Bioquímica y Biología Molecular B e Inmunología.
Facultad de Química, Regional Campus of International Excellence
“
Campus Mare Nostrum”, Universidad de Murcia, P.O. Box 4021,
E-30100 Murcia, Spain. E-mail: plozanor@um.es;
Fax: +34 868 88 41 48; Tel: +34 868 88 73 92
b
8
IEM (Institut Européen des Membranes), UMR 5635
acetate ILs, or by coating immobilized cellulase with a protec-
(
CNRS-ENSCM-UM2), Université Montpellier 2, Place E. Bataillon,
tive shell of hydrophobic ILs, like 1-butyl-3-methylimidazolium
F- 34095 Montpellier, France. Fax: +33 467149119; Tel: +33 467149148
9
bistriflimide, the identification and application of cellulases
†
1
Electronic supplementary information (ESI) available. See DOI:
0.1039/c2gc35905g
from microbial sources others than fungus (e.g., the halophilic
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10
archaeon Halorhabdus utahensis) with improved IL tolerance,
or the use of concentrated sea water as a free-IL alternative reac-
kosmotropic salt solutions after a cellulose re-precipitation step
with polar solvent to form a three-phase system forming an
11
16a
tion medium. However, the most popular approach involves
the re-precipitation of cellulose from IL solutions into polar
molecular solvents (e.g., water, ethanol, etc.) in order to disrupt
the crystalline structure of this polysaccharide, thus improving its
IL-rich phase,
or the chromatographic separation of the IL
and glucose from the enzymatic reaction mixture using alumina
as the stationary phase, which provided up to 93% IL reco-
16b
very.
However, since the final destination of the hydrolyzed
12
subsequent enzymatic hydrolysis in buffered media. The
digestibility of pretreated biomass by cellulase is limited by cel-
lulose accessibility. However, the full degradation of cellulose to
its glucose monomer units has not been reported. Some authors
have applied ultrasonic intensification during the pretreatment of
cellulose with ILs to improve the disruption of the cellulose
structure, which enhances enzymatic in situ saccharification of
cellulose in aqueous/IL media, providing up to 95% yield after
cellulose solutions is fermentation by yeast to produce bioetha-
nol, the effect of ILs on the viability of yeast cells should also be
taken into account. Recently, it has been reported how the
residual 1-ethyl-3-methylimidazolium acetate ([Emim][OAc])
content of cellulose hydrolysates act as a primary source of inhi-
bition on Saccharomyces cerevisiae growth and ethanol produc-
17a
tion.
In the same way, it was observed how an engineered
Saccharomyces cerevisiae strain, with cellulases on its cell
surface, was resistant to some cellulose-dissolving ILs (i.e.,
13
2
4 h reaction. Other authors carried out the enzymatic hydro-
−1
17b
lysis step of [Bmim][Cl]-pretreated cellulose (5 mg mL ) in a
pH 4.8 citrate buffer system containing up to 20% of another IL,
such as the tris-(2-hydroxyethyl)methylammonium methylsulfate
[Emim][OAc], [Emim][Cl], etc.) up to 200 mM.
In this context, the present work describes for the first time a
cyclic experimental approach (see Fig. 1) based on the enzymatic
hydrolysis of regenerated cellulose from [Bmim][Cl] solutions,
and the recovery and reuse of this IL in successive cellulose dis-
solution–regeneration cycles. The experimental protocol to
produce regenerated cellulose (RC) was selected as a function of
both the amount of IL recovered, and the suitability of this RC
for enzymatic hydrolysis. Thus, water, ethanol, and a 50% (mol/
mol) water–ethanol solution were tested as green antisolvents for
the precipitation of cellulose from a cellulose–[Bmim][Cl] solu-
tion, and several operation parameters, such as temperature, stir-
ring, ultrasound, etc., were assayed in an attempt to fully recover
this IL for reuse in consecutive dissolution–precipitation cycles.
−1
(HEMA), which yields up to 4 mg mL glucose after 15 h at
14
6
0 °C.
To the different advantages that IL technology may provide,
as regards cellulose processing in the bioethanol industry, should
be added the economical and environmental sustainability of the
1
b,6,7b
process based on the full recovery and recycling of ILs.
Some ILs have been described as being not fully green solvents
because of their low biodegradability and high (eco)toxicological
15
properties.
Several approaches to recover ILs from biomass–IL solutions
have recently been proposed, e.g., the use of aqueous
Fig. 1 Scheme of the cyclic protocol for the enzymatic saccharification of IL-pretreated cellulose and the recycling of the ionic liquid 1-butyl-3-
methylimidazolium ([Bmim][Cl]). RC: regenerated cellulose. For details see the Experimental section.
2632 | Green Chem., 2012, 14, 2631–2637
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2
Experimental
HPLC analysis of [Bmim][Cl]
Cellulase from T. reesei (Celluclast 1.5 L, EC 3.2.1.4), and cello-
biase from Aspergillius niger (Novozyme 188, beta-1,4-glucosi-
dase, EC 3.2.1.21) were a gift from Novozymes S.A (Spain).
Microcrystalline cellulose (20 μm powder) and other chemicals
were purchased from Sigma-Aldrich-Fluka (Madrid, Spain). The
IL 1-butyl-3-methylimidazolium chloride, [Bmim][Cl], (99%
purity) was purchased from IoLiTec GmbH (Germany).
Prior to use, enzyme preparations were ultrafiltered to elimin-
ate all the low molecular weight additives, as follows: 25 mL of
Celluclast or Novozym 188 were diluted in 225 mL of 50 mM
citrate buffer pH 4.8, and the resulting solutions were concen-
trated 10-fold by ultrafiltration at 8 °C using a Vivaflow 50
The IL concentration of all the washing fractions was determined
in a Shimadzu HPLC equipped with a multi-channel (LC-20AD)
pump and DAD (SPD-M20A) detector, using a Synergi Polar-
RP 150 × 4.6 mm column (Phenomenex) packed with polar end-
capped particles (4 μm, pore size 80 Å) as the stationary phase.
The analyses were performed under isocratic conditions
−1
(0.75 mL min flow rate) using a 70 : 30 (v/v) 5 mM phosphate
buffer (KH PO –H PO ) pH 3.0/acetonitrile as the mobile
2
4
3
4
18
phase. The elution profiles were monitored at 218 nm, and
identification and quantification of the [Bmim][Cl] peak
(2.8 min retention time) was made by the corresponding cali-
bration straight line using acetophenone (11 min retention time)
as the internal standard.
(Sartorious) system equipped with polysulphone membranes
(
10 kDa, cut-off). For each enzyme, the process was repeated
−1
three-times, leading to cellulase (0.115 U mg
1
9
protein,
Enzymatic hydrolysis of regenerated cellulose (RC)
−1
47.5 mg protein per mL) or cellobiase (0.814 U mg protein,
3.6 mg protein per mL) solutions, respectively.
The 2% (w/v) RC suspension used as the substrate was prepared
by introducing 500 mg of wet RC (80 mg dry RC) into a screw-
capped vial with a Teflon-lined septum (5 mL total capacity),
containing 4 mL of 50 mM citrate buffer pH 4.8. The mixture
was maintained under magnetic stirring in a glycerol bath at
Preparation of regenerated cellulose (RC)
Firstly, the [Bmim][Cl] (10 g) was introduced into a 100 mL
Erlenmeyer flask, and incubated at 115 °C in a thermoblock for
5
0 °C until a homogeneous suspension was observed. The reac-
tion was then started by adding both cellulase (120 μL,
1
5 min, until the IL was fully melted. Then, microcrystalline cel-
−1
1
47.5 mg prot. mL ) and cellobiase (60 μL, 93.6 mg prot.
lulose (1 g) was added, and the mixture incubated with mechan-
ical stirring for 1 h at 115 °C, which gave a clear, colourless and
viscous cellulose solution. This solution was then cooled to
−1
mL ) glycohydrolases and was magnetically stirred for 4 h. At
regular time intervals, 65 μL-aliquots were taken and suspended
in 0.1 M bicarbonate buffer, pH 9.8 (1.035 mL) to stop the reac-
tion, the samples were then centrifuged at 13 000 rpm for 5 min.
The resulting clear phase was used to quantify glucose and cello-
biose by HPLC, and the total reducing sugars by the dinitrosa-
licylic acid (DNS) method. One unit of cellulase activity was
defined as the amount of enzyme that produces 1 μmol of redu-
cing sugars per minute. One unit of cellobiase activity was
defined as the amount of enzyme that hydrolyzes 1 μmol of cel-
lobiose per minute. All experiments were carried out in
duplicate.
6
0 °C in a glycerol thermostatic bath. The cellulose was regener-
ated by adding 50 mL (approx. 5-fold IL-cellulose volume) of
water, ethanol or a equimolar (23.5 : 76.5, v/v) water–ethanol
solution, pre-heated to 60, 70 or 80 °C, and the resulting cellu-
lose suspension was vigorously stirred for 15 min. The RC gel
was recovered by filtration through a nylon membrane (0.1 mm
mesh), while the liquid fraction was collected and stored for
further analysis by HPLC and IL recovery. Then, the RC gel was
washed five times with 50 mL of different antisolvent solutions
at room temperature so that the IL was fully desorbed from the
cellulose gel. This involved two-washing steps, using the same
antisolvent as for cellulose regeneration, applying 150 W ultra-
sounds (Ultrasons, Selecta, Spain) for 15 min; two-washing
steps with ultrapure water (MilliQ–Millipore System) and mech-
anical stirring for 15 min; and a final washing step with 50 mM
citrate buffer pH 4.8 and mechanical stirring. The five resulting
washing fractions were carefully collected and stored for further
analysis by HPLC and IL recovery. The moisture content of the
resulting regenerated cellulose was 84% (w/w), as measured by
weight loss of RC after drying in an oven for 14 h at 105 °C.
Microbial growth test
Growth assays were performed using a lyophilised commercial
Saccharomyces cerevisiae strain (Maurivin-PDM, AB Mauri,
Australia) in YPD media [2% (w/v) glucose, 2% (w/v) peptone
17,19
and 1% (w/v) yeast extract].
The pH of the media were
adjusted to 6.5 before sterilising. A starter culture was first pre-
pared by adding 20 mg commercial S. cerevisiae lyophilised
powder to 40 mL standard YPD medium, which was incubated
under shaking (300 rpm) for 16 h at 30 °C in aerobic conditions.
Then, a new YPD medium (40 mL) was prepared by dissolving
peptone and yeast extract in a 2% (w/v) glucose solution in
Recovery of 1-butyl-3-methylimidazolium chloride
5
mM citrate buffer pH 4.8, previously obtained by enzymatic
The [Bmim][Cl] content of each washing liquid sample was sep-
arated by vacuum distillation at 70 °C and 74 hPa for 4 h. Then,
all the fractions containing the recovered IL were jointly intro-
duced into a 100 mL Erlenmeyer flask and incubated for 16 h in
an oven at 80 °C to dryness. The dry [Bmim][Cl] recovered was
repeatedly used to dissolve cellulose in further experiments, as
described above.
hydrolysis of RC, as described above. Prior to use, this glucose
solution was ultrafiltered using a Vivaflow 50 (Sartorious)
system equipped with polysulphone membranes (10 kDa, cut-
off) to eliminate soluble enzymes. As a growing control, another
standard YPD medium was prepared by dissolving commercial
glucose, peptone and yeast extract in 5 mM citrate buffer pH 4.8.
Then, the pH of both growing media was adjusted to 6.5 before
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a
Table 1 Effect of cellulose regeneration protocol on [Bmim][Cl] recovery as a function of antisolvent and washing step conditions
Washing steps
Entry
Cellulose precipitation with antisolvent (AS)
1. AS + US
2. AS + US
3. H
2
O
4. H
2
O
5. CB
Total IL recovery (%)
1
2
3
4
5
6
7
78.0 (H
80.0 (H
82.0 (H
2
2
2
O, 60 °C)
O, 70 °C)
O, 80 °C)
10.7
8.4
6.2
21.6
4.4
5.3
6.4
6.8
7.2
8.9
1.4
1.4
1.2
nd
nd
nd
nd
0.2
0.3
0.4
nd
nd
nd
nd
<0.1
0.1
nd
nd
nd
nd
nd
nd
nd
95.1
95.2
95.4
97.6
99.7
98.9
96.7
67.1 (EtOH, 60 °C)
93.7 (H
91.8 (H
89.9 (H
2
2
2
O–EtOH, 60 °C)
O–EtOH, 70 °C)
O–EtOH, 80 °C)
5.0
0.2
a
AS + US, antisolvent plus ultrasound for 15 min at room temperature; CB, 50 mM citrate buffer pH 4.8; nd, not detected; see Experimental section
for details of samples.
sterilising. Growth assays were run by adding 0.1 mL of the
above S. cerevisiae starter and shaking (300 rpm) at 30 °C for
respective antisolvent applied during the precipitation step, as
well as the intensification effect of ultrasounds. Then, the result-
ing RCs were washed by water (twice), and then by 50 mM
citrate buffer pH 4.8. Table 1 shows the [Bmim][Cl] recovery
yield for each step in the seven approaches tested to produce RC.
As can be seen, the use of water as the antisolvent agent to pre-
cipitate cellulose (entry 1) permitted a direct 78% IL recovery at
3
0 h in aerobic conditions. At regular intervals, the optical den-
sities (O.D.) of both cultures were determined at 600 nm
OD600) by appropriate dilution in a spectrophotometer (Novas-
(
pec II, LKB-Pharmacia).
6
0 °C, which slightly increased to 82% when the temperature
HPLC analysis of sugars
was raised to 80 °C (entries 2 and 3). In these cases, the appli-
cation of two consecutive washing steps, intensified by the
action of ultrasound, allowed improvement of the total IL recov-
ery to 95.1–95.4%. The application of additional washing steps
with water and citrate buffer did not provide further improve-
ments in IL recovery. The use of ethanol as the antisolvent
The glucose and cellobiose concentrations of the enzymatic reac-
tion samples were determined in a Shimadzu HPLC equipped
with a multi-channel (LC-20AD) pump, oven and light scatter-
ing (ELSD-LT II) detector. A Rezex RCM-monossacharide-Ca
column (300 × 7.8 mm, Phenomenex) was used as the stationary
+2
(entry 4) provided the worst results during direct cellulose pre-
phase at 60 °C. Analyses were performed in isocratic conditions
cipitation at 60 °C (67.1% IL yield), which rose to 97.5% after
two consecutive washing steps with the same antisolvent and
intensification by the action of ultrasound. Once again, this yield
was not improved by additional washing steps with both water
and citrate buffer. Higher temperatures were not assayed because
of the boiling point of ethanol (78 °C). However, the best results
were obtained by using the equimolar ethanol–water mixture as
the antisolvent at 60 °C (entry 5), resulting directly in a 93.7%
IL recovery yield. The increase in temperature during the precipi-
tation step produced a decrease in the IL recovery yield (entries
6 and 7). The application of the proposed washing protocol to
the resulting RC samples improved the total IL recovery yield up
to 99.7% (entry 5), clearly demonstrating the suitability of the
proposed methodology to reach full recovery of ILs in cellulose
processing. These results cannot be explained simply by the
ability of either the water or ethanol protic solvents to shift
[Bmim] and [Cl] ions from the RC, because neither is able to
achieve full recovery when used as pure solvent (entries 1 to 4).
In this context, Lindman et al. suggested that cellulose is signifi-
cantly amphiphilic and that the hydrophobic interactions are
1
(
0.6 mL min− flow rate) using water as a mobile phase. The
glucose (11 min retention time) and cellobiase (9 min retention
time) peaks were identified and quantified from the correspond-
ing calibration straight lines, using xylitol (23 min retention
time) as the internal standard.
3
Results and discussion
The regeneration of cellulose from IL solutions is a key step in
weakening its crystallinity and/or disrupting its fibre organiz-
ation, resulting in a more accessible substrate for enzymatic
hydrolysis into fermentable sugars, and subsequent transform-
1
2
ation into bioethanol. However, the recovery and reuse of ILs
in cellulose processing for the biofuel industry could be con-
sidered as an essential task for ensuring the sustainability of any
proposed approach in this field. Taking into account that
[Bmim][Cl] has been described as one of the best ILs for dissol-
4
,5
ving lignocellulosic materials, a 10% cellulose solution in this
IL was chosen as starting material to prepare RC, while water,
ethanol or an equimolar water–ethanol mixture, were used as
antisolvents to precipitate cellulose. In preliminary experiments,
it was observed how the RC texture was hard and lumpy when
the precipitation step was carried out at room temperature, while
a fine and soft RC powder was obtained at higher temperatures,
i.e., 60, 70 or 80 °C. Seven different experimental protocols
were followed to produce RC, and each RC sample was washed
five times in order to attain full IL recovery (see Fig. 1). For all
RC cases, the first two washing steps were carried out using the
20
important for explaining its solubility pattern. Thus, the full IL
recovery obtained for the equimolar water–ethanol mixture could
be explained by both the role of water and ethanol as very strongly
hydrogen-bonded molecules, as well as, the hydrophobic proper-
ties of the aliphatic moiety of the ethanol molecule, behaving
together a useful green solvent for IL recovery. Furthermore, the
role of ultrasound in the two washing steps could also be con-
sidered as the key, because of the demonstrated efficiency for both
13,21
the disruption and dissolution of cellulose fibres.
2634 | Green Chem., 2012, 14, 2631–2637
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Fig. 3 Time-course profiles of total reducing sugars (○), glucose (●)
and cellobiase (▲) released by the combined action of cellulase and cel-
lobiase, using the regenerated cellulose (RC) resulting from precipitation
with water–ethanol (see Table 1, entry 5) as substrate.
as fast as it was formed. The suitability of this RC substrate for
enzymatic hydrolysis was also evident from the change in turbid-
ity observed in the reaction medium (see inset pictures in Fig. 3),
measured by transmittance at 660 nm, which was shifted from 0
to 70% during the 4 h the reaction. These results are clearly
related by the weakening of the cellulose crystallinity and disrup-
tion of the fibre organization produced by the dissolution/precipi-
tation pretreatment, which resulted in a more accessible substrate
for efficient enzymatic hydrolysis. However, full enzymatic
hydrolysis of the RC substrate to its glucose monomeric units
could only be explained by the added value made possible by
the full recovery of the [Bmim][Cl] during the cellulose pretreat-
ment process.
Fig. 2 Time-course profiles of both cellulose hydrolysis degree, and
total reducing sugars released by the combined action of cellulase and
cellobiase using regenerated cellulose (RC) as substrate. The different
RCs were obtained from 2% (w/w) cellulose solutions in [Bmim][Cl]
using water or ethanol (A), or a 1 : 1 (mol : mol) ethanol–water solution
11
(B), as antisolvents at different temperatures. The control reaction was
carried out using microcrystalline cellulose as substrate (see details in
the Experimental section).
The negative effect of all ILs, that are able to dissolve cellu-
lose, on the enzymatic activity of cellulase has been widely
Besides the advantage of recovering IL, industrial interest for
any such experimental approach will also be based on the suit-
ability of the resulting cellulosic substrate for enzymatic degra-
dation to produce glucose solutions. Fig. 2 shows the time
course profiles for the total reducing sugars produced by the
combined action of both cellulase and cellobiase on the resulting
RC substrates obtained using the protocols described in Table 1.
A control reaction was also carried out by using microcrystalline
cellulose as substrate. As can be seen, all the RCs were suitable
substrates for enzymatic hydrolysis, which was practically com-
plete after 4 h. The positive effect of [Bmim][Cl] pretreatment
on the disruption of the cellulose structure can be clearly
observed by comparison with the reaction control, where the
enzymatic hydrolysis of microcrystalline cellulose stopped at
7,8,10,12,13
7b
described.
For the case of both the [Bmim][Cl] and
7
c
1,3-dimethylimidazolium dimethylphosphate (Mmim][DMP])
ILs, it was reported that the cellulase activity decays continu-
ously with increasing IL concentration in the reaction medium,
resulting in a residual activity of up to 35–45% at 10% IL con-
centration. Furthermore, it was described how the cellulose
hydrolysis degree was clearly reduced by the presence of residual
IL (e.g., 10% [Mmim][DMP]), giving a 40–70% hydrolysis
7
c
yield at 24 h and did not increase with longer reaction times.
The use of “compatible” IL–cellulase systems (e.g., 15%
[Emim][AcO]) for enzymatic hydrolysis of cellulosic biomass
did not improve its conversion to glucose and cellobiose, giving
8d
only a 40–50% hydrolysis yield for the first 24 h. The presence
of residual IL molecules bound to the cellulose polymer may
have been involved in the low efficiency of cellulase in reaching
full saccharification. Thus, the washing intensification steps
carried out on regenerated cellulose (see Table 1) can be
regarded as being the perfect substrate conditioning step for
enzymatic hydrolysis, rather than an IL recovery process. The
regenerated cellulose, that is to be used for enzymatic saccharifi-
cation, should be fully free of residual IL content to reach full
transformation to glucose monomeric units.
5
8%. No further improvements in cellulose hydrolysis were
observed even after a reaction time of 24 h. Furthermore, the
suitability of these RCs, used as substrates for enzymatic
hydrolysis, can also be observed in Fig. 3. By using the RC
obtained with water–ethanol as antisolvent (see Table 1, entry
5
), the enzymatic hydrolysis of this RC substrate showed excel-
lent agreement between all the time course profiles of total redu-
cing sugars, glucose and cellobiose into the reaction medium.
Cellobiose was only observed at the beginning of the reaction
course, when the RC concentration was high, after which the
synergic action of cellulase and cellobiase hydrolysed cellobiose
Despite the great interest of recovering all the IL used for con-
ditioning cellulose for subsequent enzyme catalysis, its reuse in
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Fig. 5 Recovery yield of [Bmim][Cl] during consecutive operation
cycles for cellulose dissolution/regeneration by using either water (black
bars), or 1 : 1 (mol : mol) ethanol–water (white bars), as the antisolvent
agent in the precipitation step.
Fig. 4 Time-course profiles of both cellulose hydrolysis degree, and
total reducing sugars released by the combined action of cellulase and
cellobiase on regenerated cellulose (RC). The RCs were obtained
through consecutive cycles of recovery and reuse of the [Bmim][Cl]
used to dissolve cellulose, and by using water at 80 °C (A), or 1 : 1
(mol : mol) ethanol–water at 60 °C (B), as antisolvents during the cellu-
lose regeneration step.
Fig. 6 Aerobic growth of S. cerevisiae in YPD medium containing 2%
(
w/v) glucose solution at pH 6.5 and at 30 °C. The glucose was obtained
the further dissolution/precipitation of cellulose is a key factor
for the economic sustainability of the proposed technology for
biomass processing to produce biofuels. In this way, the IL
recovered by using either water or an equimolar water–ethanol
mixture, as antisolvent (see Table 1, entries 3 and 5, respect-
ively) were applied in iterative cycles of RC production. The
suitability of the resulting amorphous celluloses to be hydrolysed
by the combined action of cellulase and cellobiase can be
observed in Fig. 4, where the time course profiles for total redu-
cing sugars are depicted. As can be seen, the reuse and recycling
of [Bmim][Cl] for 5 cycles in the production of RC provided an
excellent substrate for enzymatic hydrolysis, because the
maximum conversion yield (higher than 96% for all cases) was
reached at 4 h of reaction. The ability of [Bmim][Cl] to dissolve
cellulose remained unchanged for 5 cycles, despite slight dar-
kening, which could be removed by using charcoal. Fig. 5 shows
the [Bmim][Cl] recovery yield obtained after each cellulose dis-
solution/precipitation cycle using either water or equimolar
water–ethanol mixture as antisolvent. As can be seen, the suit-
ability of the proposed methodology is clearly demonstrated by
the excellent recovery yield (higher than 93%) obtained in all
cases. The slight decrease observed as the number of cycles
increases can be attributed to handling errors during manipu-
lation of the large number of washing liquid fractions. Further-
by enzymatic hydrolysis of free-IL RC (▲), and from commercial sup-
pliers (●).
shows any sign of the decomposition and/or contaminating side
products (see ESI†) with respect to the standard IL, which
clearly demonstrates the excellent suitability of the proposed
protocol.
The suitability of the glucose solution resulting from the enzy-
matic hydrolysis of RC was also tested as a carbon source for
growing Saccharomyces cerevisiae. A YPD medium for growing
microbes was prepared by adding the corresponding amount of
peptone and yeast extract to a 2% glucose solution in 5 mM
citrate buffer pH 4.8, previously obtained by the enzymatic
hydrolysis of IL-free RC (see Experimental section), being com-
pared with a control YPD medium containing commercial
glucose. The citrate buffer concentration was ten-times lower
than the usual concentration used for enzymatic activity assays
to minimize its role as an alternative carbon source for growing
yeast. In both YPD cases, the pH of the medium was adjusted to
6.5 before inoculation. Fig. 6 shows the aerobic growth of S. cer-
evisiae in both YPD media at 30 °C. As can be seen, both time-
course profiles of microbial growth were identical, which clearly
demonstrates the excellent suitability of the glucose solution
obtained from the enzymatic reaction media for RC hydrolysis.
1
more, the H-NMR analysis of the recovered [Bmim][Cl] did not
2636 | Green Chem., 2012, 14, 2631–2637
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These results can be directly attributed to the absence of residual
IL in the RC substrate, the final glucose solution showing identi-
cal suitability to the commercial one for microbial transform-
ation. The negative effect of the residual content of IL in
S. cerevisiae growing media has been reported as a primary
source of inhibition on downstream microbial growth and
(c) S. Bose, D. W. Armstrong and J. W. Petrich, J. Phys. Chem. B, 2010,
114, 8221–8227; (d) M. E. Zakrzewska, E. Bogel-Lukasik and R. Bogel-
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(a) P. Lozano, Green Chem., 2010, 12, 555–569; (b) P. Dominguez de
María and Z. Maugeri, Curr. Opin. Chem. Biol., 2011, 15, 220–225;
6
7
(c) P. Lozano, E. Garcia-Verdugo, S. V. Luis, M. Pucheault and
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16
ethanol production. It should be noted at this point, how the
reported approaches for IL separation from monosaccharides did
not lead to full recovery of the IL. Thus, in agreement with the
(a) M. B. Turner, S. K. Spear, J. G. Huddleston, J. D. Holbrey and
R. D. Rogers, Green Chem., 2003, 5, 443–447; (b) A. C. Salvador,
M. C. Santos and J. A. Saraiva, Green Chem., 2010, 12, 632–635;
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2
2
first principle of green chemistry, dealing with prevention, it is
better (and easier) to recover ILs from RC than to treat or clean
up the resulting glucose solution from the RC hydrolysis step.
In conclusion, this work has shown how the suitability of IL
technology in biomass processing to provide useful substrates
for enzymatic hydrolysis is clearly enhanced by the IL recovery.
By combining green molecular antisolvents, like water or
ethanol, and ultrasound, efficient protocols to fully recover the
IL from cellulose solutions can be designed. Moreover, the IL
can be purified by simple distillation, before being applied in
further biomass processing. In addition to the economic and
environmental benefits provided by the full recovery of IL, the
excellent suitability of the resulting amorphous cellulosic sub-
strate for full transformation into directly fermentable glucose
should be emphasized. Fundamental studies on enzyme reactors
need to be carried out to establish clear criteria for specifically
pairing the most appropriate enzyme mixture with the corre-
sponding pretreated biomass substrate in continuous operation.
Once again, the IL technology appears as an enhancer of
enzyme technology for developing green chemical bioprocesses.
This work was partially supported by MINECO CTQ2011-
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