Aqueous ionic liquids and deep eutectic solvents for cellulosic biomass pretreatment and saccharification

Article abstract of DOI:10.1039/c3ra46149a

Ionic liquids (ILs) have proven effective solvents for pretreating lignocellulose, leading to the fast saccharification of cellulose and hemicellulose. However, the high cost of most ILs remains a major barrier to commercializing this recent approach at a practical scale. As a strategic detour, aqueous solutions of ILs are also being explored as less costly alternatives to neat ILs for cellulose pretreatment. However, limited studies on a few select IL systems are known and there remains no systematic survey of various ILs, eluding an in-depth understanding of pretreatment mechanisms afforded by aqueous IL systems. As a step toward filling this gap, this study presents results for Avicel cellulose pretreatment by neat and aqueous solutions (1.0 and 2.0 M) of 20 different ILs and three deep eutectic solvents, correlating enzymatic hydrolysis rates of pretreated cellulose with various IL properties such as hydrogen-bond basicity, polarity, Hofmeister ranking, and hydrophobicity. The pretreatment efficiencies of neat ILs may be loosely correlated to the hydrogen-bond basicity of the constituent anion and IL polarity; however, the pretreatment efficacies for aqueous ILs are more complicated and cannot be simply related to any single IL property. Several aqueous IL systems have been identified as effective alternatives to neat ILs in lignocellulose pretreatment. In particular, this study reveals that aqueous solutions of 1-butyl-3-methylimidazolium methanesulfonate ([BMIM][MeSO ₃]) are effective for pretreating switchgrass (Panicum virgatum), resulting in fast saccharification of both cellulose and hemicellulose. An integrated analysis afforded by X-ray diffraction, scanning electron microscopy, thermogravimetric analysis and cellulase adsorption isotherm of lignocellulose samples is further used to deliver a more complete view of the structural changes attending aqueous IL pretreatment.

Full text of DOI:10.1039/c3ra46149a

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Aqueous ionic liquids and deep eutectic solvents
for cellulosic biomass pretreatment and
saccharification†
Cite this: RSC Adv., 2014, 4, 10586
a
b
a
b
c
*
Shuqian Xia, Gary A. Baker, Hao Li, Sudhir Ravula and Hua Zhao
Ionic liquids (ILs) have proven effective solvents for pretreating lignocellulose, leading to the fast
saccharification of cellulose and hemicellulose. However, the high cost of most ILs remains a major
barrier to commercializing this recent approach at a practical scale. As a strategic detour, aqueous
solutions of ILs are also being explored as less costly alternatives to neat ILs for cellulose pretreatment.
However, limited studies on a few select IL systems are known and there remains no systematic survey
of various ILs, eluding an in-depth understanding of pretreatment mechanisms afforded by aqueous IL
systems. As a step toward filling this gap, this study presents results for Avicel cellulose pretreatment by
neat and aqueous solutions (1.0 and 2.0 M) of 20 different ILs and three deep eutectic solvents,
correlating enzymatic hydrolysis rates of pretreated cellulose with various IL properties such as
hydrogen-bond basicity, polarity, Hofmeister ranking, and hydrophobicity. The pretreatment efficiencies
of neat ILs may be loosely correlated to the hydrogen-bond basicity of the constituent anion and IL
polarity; however, the pretreatment efficacies for aqueous ILs are more complicated and cannot be
simply related to any single IL property. Several aqueous IL systems have been identified as effective
alternatives to neat ILs in lignocellulose pretreatment. In particular, this study reveals that aqueous
solutions of 1-butyl-3-methylimidazolium methanesulfonate ([BMIM][MeSO3]) are effective for
pretreating switchgrass (Panicum virgatum), resulting in fast saccharification of both cellulose and
hemicellulose. An integrated analysis afforded by X-ray diffraction, scanning electron microscopy,
thermogravimetric analysis and cellulase adsorption isotherm of lignocellulose samples is further used to
deliver a more complete view of the structural changes attending aqueous IL pretreatment.
Received 27th October 2013
Accepted 18th December 2013
DOI: 10.1039/c3ra46149a
www.rsc.org/advances
pervasive use of the currently expensive imidazolium cations in
designing cellulose-dissolving ILs.
1 Introduction
To address the high cost issue of ILs, a number of different
approaches have been considered in addition to the recycling
and reuse of ILs.6–8 The rst approach entails the synthesis of
some novel but less expensive ILs for cellulose dissolution. Along
these lines of inquiry, our group has recently prepared relatively
inexpensive and low-viscosity ether-functionalized alkylammo-
nium and piperidinium ILs bearing acetate anions that are able
to dissolve ꢁ10 wt% cellulose.9,10 Other groups have prepared
cellulose-dissolving ILs containing amino acid-derived anions:
(a) [EMIM][glycinate] reportedly afforded the complete dissolu-
tion of bamboo biomass,11 and (b) N,N-diethyl-N-(2-methoxy-
ethyl)-N-methylammonium ILs containing amino acid anions
like alaninate were able to dissolve 5–12 wt% cellulose at 100 ꢀC.12
King et al.13 prepared novel guanidine-based ‘distillable’ acid–
base conjugate ILs for dissolving microcrystalline cellulose (up to
5–10 wt%) at 100 ꢀC. Zhang et al.14 developed a new CO2-triggered
switchable system by mixing DMSO, non-ionic bases, and
CO2. This switchable solvent platform was able to dissolve up to
15 wt% microcrystalline cellulose; the regenerated cellulose
displayed a reduced crystallinity and a fast hydrolysis rate.
Ionic liquids (ILs) are unconventional, essentially non-volatile
ionic organic solvents with melting points generally below 100
ꢀC. Some ILs are capable of dissolving cellulosic biomass and
have thus become the subject of intensive study as alternative
media for lignocellulose pretreatment toward bioethanol
production.1–3 Although IL pretreatment oen leads to an
accelerated saccharication of cellulosic biomass, this method
has encountered several obstacles associated with ILs such as
their high cost, energy intensive recycling, and environmental
concerns over their toxicity and biodegradability.4,5 It is worth
noting that the high IL cost is at least in part due to the
aSchool of Chemical Engineering & Technology, Tianjin University, Tianjin 300072,
China
bDepartment of Chemistry, University of Missouri-Columbia, Columbia, MO 65211,
USA
cDepartment of Chemistry and Forensic Science, Savannah State University, Savannah,
GA 31404, USA. E-mail: huazhao98@gmail.com; zhaoh@savannahstate.edu; Tel: +1-
912-358-4448
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ra46149a
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A second strategy for improving the economics of IL-based addition, although the cellulose dissolution capability of neat
cellulose pretreatment is substituting neat ILs with ionic or ILs is oen associated with the hydrogen-bond basicity of their
eutectic mixtures. For example, the Jerome group15 claimed that constituent anions,9,24 it is not currently understood how
´ ˆ
inexpensive ionic mixtures of cholinium acetate with 5–15 wt% aqueous ILs impact biomass pretreatment, as cellulose is
of tributylmethylammonium chloride could dissolve 2–6 wt% insoluble in known aqueous IL systems.
microcrystalline cellulose (i.e., Avicel PH 105) at 110 ꢀC.
Aiming at an in-depth understanding of cellulose pretreat-
Rinaldi16 reported that amide-related solvents (such as 1,3- ment by aqueous ILs, we set out to determine and compare
dimethyl-2-imidazolidinone and DMSO) containing a small saccharication rates for cellulose pretreated with 20 different
fraction of [EMIM][OAc] or [BMIM]Cl (at molar fractions below ILs and three cholinium-based DESs, neat and at 1.0 and 2.0 M
0.3, for instance) could instantaneously dissolve 10 wt% cellu- concentrations in water. In order to better understand the
lose at 100 ꢀC. In another example, the Francisco group17 pretreatment process, we also examined the cellulosic struc-
designed biorenewable deep eutectic solvents (DESs) based ture by X-ray diffraction (XRD), scanning electron microscopy
on betaine, choline, and amino acids coupled with lactic or (SEM), and thermogravimetric analysis (TGA), as well as
malic acid for dissolving large amounts (up to 12 to 15 wt%) of attempting to correlate the enzymatic hydrolysis rate with the
lignin (despite the poor solubility of cellulose), and further physical properties of the ILs. Finally, we also tested the
demonstrated the utility of their DESs in the delignication of saccharication efficiency for switchgrass pretreated with neat
wheat straw.
and aqueous ILs. The mechanistic insights offered by our
A third approach used to minimize the cost associated with systematic study bring into focus a clearer picture of what
using ILs to treat cellulosic materials involves the addition of future role aqueous ILs might play in lignocellulose pretreat-
compatible organic co-solvents. Unlike solvents such as alco- ment and saccharication.
hols, the co-solvents selected for this application are those that
do not precipitate cellulose from the IL solution, at least within
the chosen solvent composition range. In recent work, the
Heinze group18 systematically examined the impact of 18 2.1 Materials
2 Experimental
solvents and three binary solvent mixtures on cellulose solu-
tions in IL and indicated that ideal co-solvents (such as DMSO,
A number of ILs were acquired from commercial sources,
although several were prepared in house (see Table S1 in (ESI†)
for details). The following chemicals and enzymes were
purchased from Sigma-Aldrich (St. Louis, MO): Avicel PH-101
cellulose (microcrystalline cellulose (MCC), particle size
50 mm, DP 225, produced by FMC Corp.), cellulase from Tri-
DMF and dichloromethane) typically have solvatochromic
polarity ENT values above 0.3, very low ‘acidity’ (a < 0.5), and
relatively high ‘basicity’ (b $ 0.4). The use of these co-solvents
not only forms homogeneous cellulose solutions in ILs, but also
considerably reduces the cost and viscosity of the resulting IL-
based system. Tian et al.19 examined the enzymatic digestibility
of microcrystalline cellulose pretreated by organic electrolyte
solutions (mixtures of 1-allyl-3-methylimidazolium chloride
choderma reesei (product # 22173, lot # 1086305, E.C. #3.2.1.4,
slightly brown powder, 6.7 U mgꢂ1 or 0.5 FPU mgꢂ1) and
glucose (HK) assay kit. b-Glucosidase from sweet almonds was
obtained from MP biomedicals (product
# 100348, lot
#R28034, 3811 U mgꢂ1‡). Switchgrass was a kind gi from Dr
H. M. O'Neill (Oak Ridge National Laboratory); it was grown,
harvested, dried, and then knife-milled to an average particle
size of 0.5 mm. D-Xylose assay kit (catalog # K-XYLOSE) was
obtained from the Megazyme International Ireland Ltd. (Bray,
Co. Wicklow, Ireland).
([AMIM]Cl) with DMSO) and suggested that the hydrolysis yield
and rate increased with the mole fraction of [AMIM]Cl in the
mixture; aer cellulose pretreatment by an organic electrolyte
solution containing 0.7 mole fraction of [AMIM]Cl, they ach-
ieved 54.1% glucose yield, which was 7.2 times higher than that
of untreated cellulose.
Yet a fourth approach concerns pretreating cellulose with
aqueous solutions of ILs (i.e., as opposed to neat ILs). For
example, the Mazza group20,21 achieved an optimum ferment-
able sugar recovery of 71.4% from wheat straw when using an
2.2 Pretreatment of Avicel PH-101 and switchgrass by IL
solutions
ꢀ
aqueous solution of 49.5 wt% [EMIM][OAc] at 158 C for 3.6 h.
Most attempts at IL pretreatment of cellulosic biomass can be
subsumed under two broad categories: regeneration pretreat-
ment and suspension pretreatment. Both avenues were involved
in the course of the current work. (1) Regeneration pretreatment.
When employing neat cellulose-dissolving ILs, 2.0 mL of an IL
within a glass test tube was immersed in an oil bath held at
Brandt et al.22 obtained ca. 90% glucose yields from cellulosic
biomass pretreated by aqueous solutions of 80 volꢀ.% [BMIM]-
[MeSO3], [BMIM][MeSO4], or [BMIM][HSO4] at 120 C for 22 h,
but observed lower saccharication yields aer pretreatment
with 80 vol.% acetate-based ILs. The Zhang group23 pretreated
ꢀ
ꢀ
sugarcane bagasse at 130 C for 30 min by aqueous [BMIM]Cl
110 C. Samples of cellulose were carefully added to the IL in
solution containing 1.2% HCl, leading to a glucan recovery of
>90% and digestibility of 94–100% aer 72 h of enzymatic
hydrolysis; however, the acidic condition resulted in the loss of
most xylan. Although these aqueous ILs appear promising
alternatives to neat ILs for lignocellulose pretreatment, there
10 mg increments. Aer each addition step, a clear and visually
homogeneous solution was awaited before proceeding to the
next addition. The cellulose was incubated in the oil bath for 4 h
‡ In this case, 1 U corresponds to the liberation of 1.0 mg of glucose per minute at
currently exists no study screening a wide spectrum of ILs. In 35 ꢀC.
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ꢀ
at 110 ꢀC. Aer cooling to room temperature, the cellulose was was stirred continuously at 4 C. Aer 30 min, the suspension
regenerated by the addition of distilled water followed by solution was centrifuged to settle the insoluble cellulose. Thirty
vigorous stirring for 30 min. The ensuing cellulose precipitates microliters (30 mL) of the supernatant were added into a curvet
were collected by vacuum ltration, followed by thorough containing 1.5 mL of Bradford reagent. Aer mixing the solu-
washing and drying using distilled water followed by acetone. tion at room temperature for 10 min, the protein concentration
The pretreated cellulose was dried in air for 48 h before use in was determined by its absorbance at 595 nm (blank was 30 mL
experiments. (2) Suspension pretreatment. In cases where IL citrate buffer in 1.5 mL Bradford reagent). A protein calibration
solutions could not fully dissolve cellulose, 0.2 g of cellulose (or curve was generated from standard solutions of bovine serum
0.1 g of switchgrass) was suspended in 2.0 mL of an IL (or its albumin (BSA). The amount of adsorbed protein on cellulose
aqueous solution) and incubated in an oil bath at 110 ꢀC for 4 h. was calculated from subtracting the remaining protein in
Aer the solution was cooled to room temperature, distilled solution aer 30 min from the initial protein content.
water was added into the solution with vigorous stirring for 30
min. The treated cellulose was collected, washed, and dried
exactly as written for the regeneration pretreatment approach.
3 Results and discussion
3.1 Screening of ILs for cellulose pretreatment and the
hydrolysis rate model
2.3 Enzymatic hydrolysis of cellulose and switchgrass
In this work, we chose Avicel PH-101 as our model cellulose
A suspension of 20 mg (untreated or pretreated) of Avicel PH-
because it represents a well-studied microcrystalline cellulose
101 cellulose or switchgrass (2% wt/v loading) in 1.0 mL of
(MCC). We tested a wide variety of ionic solvent systems for
citrate buffer solution (50 mM, pH 4.8) was incubated in a water
cellulosic pretreatment, including 20 different ILs and three
cholinium-based deep eutectic solvents (see Table S1 in ESI†) in
Avicel cellulose pretreatment; the ionic solvents employed were
either neat (anhydrous) or diluted in water to 1.0 or 2.0 M
bath at 50 ꢀC under gentle stirring. The reaction was initiated by
adding 3.0 mg (or 20 U) of Trichoderma reesei cellulase and 1.0
mg (or 3811 U) of b-glucosidase. An aliquot of 50 mL of the
reaction mixture was periodically withdrawn and diluted with
concentrations. Among those ILs examined, ones containing
citrate buffer. The glucose concentration was determined by the
the chloride, acetate, or dimethylphosphate anion (viz., entries
glucose HK assay method at 340 nm using a Shimadzu UV-Mini-
1, 5, 13, and 16–20 in Table 1) appeared most adequate for
dissolving appreciable amounts of cellulose (typically, 5–
10 wt%). We further carried out enzymatic hydrolysis on native
Avicel PH-101 or cellulose pretreated with the 23 different ionic
1240 spectrophotometer.25 The xylose concentration was deter-
mined by the D-xylose assay kit spectrophotometrically at 340
nm.26 All experiments were run in duplicate.
solvent systems (neat and as 1.0 and 2.0 M solutions in water; 69
total solvent systems); typical hydrolysis kinetics proles are
2.4 XRD And SEM characterization
illustrated in Fig. S1 (ESI†) using the example of [BMIM][OAc].
As shown in Fig. S1,† the hydrolysis of cellulose regenerated
from neat [BMIM][OAc] was much faster that cellulose pre-
treated by aqueous [BMIM][OAc] solutions.
XRD patterns for untreated and IL-pretreated Avicel celluloses
were analyzed at 25 C by a D8-Focus X-ray diffraction system
ꢀ
˚
(Bruker AXS GmbH) using CuKa radiation (l ¼ 1.542 A) gener-
ated at 40 kV and 40 mA. Samples were scanned over the
angular range 2q ¼ 6–45ꢀ, with a step size of 0.02ꢀ and a step
time of 0.5 s. The morphologies of the cellulosic materials, with
or without IL pretreatment, were examined by scanning electron
microscopy (S-4800, Hitachi, Ltd, Japan) at a 3 kV accelerating
voltage.
To quantitatively compare the hydrolysis rates, a simple
Michaelis–Menten model was used to evaluate the hydrolysis
reaction rate. Although cellulase is a cocktail consisting of
several major enzymes (such as endoglucanase, exoglucanase
and b-glucosidases), many studies have suggested that the
Michaelis–Menten equation (competitive or noncompetitive
inhibition)27–29 or its modications (such as kinetics with
competitive inhibition and Langmuir adsorption)30,31 could
describe the kinetics of enzymatic hydrolysis of cellulose.
Therefore, this study adopts the Michaelis–Menten equation
(eqn (1)) to curve-t our hydrolysis data.
2.5 Thermogravimetric analysis
Thermogravimetric analysis (TGA) was performed with a TA
Instruments TGA Q50 or TGA Q500 under a nitrogen atmo-
sphere (40 mL minꢂ1) using Pt pans and scanning from 20 to
600 ꢀC at a heating rate of 10 ꢀC minꢂ1. The decomposition
temperature Tdcp denotes the thermal onset of decomposition,
signifying the occurrence of a 10% total mass loss during TGA
scanning. Tder is another measure of decomposition tempera-
ture, which is dened as the maximum in the rst-derivative
envelope of the TGA scan.
d½Sꢃ
dt
Vmax½Sꢃ
¼ ꢂ
(1)
Km þ ½Sꢃ
Here, [S] is the concentration of substrate (i.e. cellulose)
which can be estimated from the initial cellulose concentration
(20 g Lꢂ1) and time-dependent glucose concentration (deter-
mined by the glucose HK assay periodically); Vmax represents
the maximum hydrolysis rate; Km is the substrate concentration
2.6 Cellulase adsorption isotherm
A known amount of cellulase was dissolved in 1.0 mL citrate at which the reaction rate is half of Vmax; Vmax/Km represents the
ꢀ
buffer (pH 4.8, 50 mM) at 4 C. The cellulose powder (20 mg) reaction rate constant at a low substrate concentration. The
was added into the enzyme solution. The suspension solution Runge–Kutta algorithm (see ESI†) was used in the curve-tting
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Table 1 Effect of pretreatment by ionic solvents on the hydrolysis rate of cellulosea
Vmax/Km (g Lꢂ1 hꢂ1) and (R2)b
Neat
Ionic solvent
2.0 M
1.0 M
None (untreated)
0.10 (0.984)
Different anions
1
2
3
4
5
6
7
[BMIM]Cl
[BMIM]Br
0.22c (0.972)
0.12d (0.989)
0.11 (0.985)
0.11 (0.998)
0.46 (0.980)
0.065 (0.991)
0.093 (0.988)
0.036 (0.978) (4.0 M)
0.13 (0.980)
0.11 (0.989)
0.13 (0.988)
0.12 (0.995)
0.42 (0.990)
0.088 (0.977)
0.13 (0.983)
0.12(0.990)
0.13 (0.991)
0.13 (0.996)
0.10 (0.991)
0.13 (0.976)
0.078 (0.994)
0.16 (0.985)
0.081 (0.968)
0.14 (0.996)
0.10 (0.987)
0.11 (0.988)
0.11 (0.994)
0.12 (0.987)
0.10 (0.976)
0.13 (0.960)
0.15 (0.980)
0.11 (0.993)
0.12 (0.992)
0.10 (0.991)
0.14 (0.971)
0.083 (0.975)
0.14 (0.985)
0.090 (0.975)
0.14 (0.989)
0.11 (0.995)
0.12 (0.984)
0.12 (0.993)
0.12 (0.966)
0.13 (0.989)
0.13 (0.982)
[BMIM][BF4]
[BMIM][CF3COO]
[BMIM][OAc]
[BMIM][OTf]
[BMIM][MeSO3]
[BMIM][HSO4]
[BMIM][SCN]
[BMIM][dca]
[BMIM][NO3]
[BMIM][MeSO4]
[BMIM][Me2PO4]
[BMIM][PF6]
[BMIM][Tf2N]
8
9
10
11
12
13
14
15
Different cations
16
5
17
18
19
20
[EMIM][OAc]
[BMIM][OAc]
[HMIM][OAc]
[CH3(OCH2CH2)3–Et-Im][OAc]
[CH3(OCH2CH2)2–Et3N][OAc]
[CH3(OCH2CH2)3–Et-Pip][OAc]
0.44 (0.927)
0.46 (0.980)
0.21 (0.974)
0.25 (0.995)
0.34 (0.955)
0.40 (0.960)
0.14 (0.970)
0.13(0.976)
0.13 (0.984)
0.10 (0.981)
0.13 (0.998)
0.10 (0.960)
0.12 (0.992)
0.14 (0.971)
0.13 (0.992)
0.12 (0.990)
0.13 (0.991)
0.15 (0.985)
Deep eutectic solvents (DES)
21
22
23
Choline chloride/urea (1 : 2)
Choline chloride/glycerol (1 : 2)
Choline acetate/glycerol (1 : 1.5)
0.085 (0.998)
0.13 (0.950)
0.10 (0.982)
0.10 (0.988)
0.11 (0.986)
0.11 (0.987)
0.10 (0.967)
0.14 (0.997)
0.13 (0.970)
a
Hydrolysis conditions: 1.0 mL citrate buffer (pH 4.8, 50 mM), 0.02 g untreated or pretreated Avicel PH-101, 3.0 mg Trichoderma reesei cellulase and
b
1.0 mg b-glucosidase under gentle agitation at 50 ꢀC. Calculated from hydrolysis data using the Michaelis–Menten equation (the number in
parenthesis represents R2). Italicized data indicate regeneration pretreatment and others are suspension pretreatments; bold Vmax/Km values
c
d
indicate signicantly high hydrolysis rates. Cellulose was partially dissolved in neat IL.
analysis with the Microso Excel GRG nonlinear method to than untreated cellulose (0.10 g Lꢂ1 hꢂ1). As has been explained
obtain the Vmax and Km values for each hydrolysis reaction in earlier studies, cellulose regenerated from an IL is more
within 5 h. The Vmax/Km values are compiled in Table 1. The R2 accessible to cellulase action due to the reduction in crystal-
values are in the range of 0.927–0.998 and most of them fall linity, a transition from cellulose I to cellulose II structure, as
between 0.97 and 0.99, which suggests the simple the Michae- well as other factors.10,32–34 In this study, we are interested in
lis–Menten equation correlates the hydrolysis data quite well. learning how different anions and cations within neat ILs, as
The results for the ionic solvents shown in Table 1 are divided well as aqueous IL solutions, inuence cellulose accessibility
into three distinct categories: (i) ILs based on the [BMIM]+ and hydrolysis. In order to facilitate a general discussion of
cation with different anions, (ii) ILs based on the acetate (OAcꢂ) trends in the anion's inuence on cellulose hydrolysis rates, we
anion paired with different cations, and (iii) deep eutectic visualize our results for a series of [BMIM]+ ILs in Fig. 1. Broadly,
solvents (DESs). As a benchmark, untreated Avicel showed a for each ionic solvent condition (i.e., neat, 1.0 M (aq), and 2.0 M
Vmax/Km value of 0.10 g Lꢂ1 hꢂ1, and this forms the basis for (aq)) we can categorize the ILs into three groups: (i) those
comparing how various IL-pretreatments inuence cellulose achieving hydrolysis rates for treated Avicel much faster than
hydrolysis.
that observed for untreated Avicel, (ii) those reaching only
slightly faster rates, and (iii) those showing a similar hydrolysis
rate as the untreated cellulose.
3.2 Impact of IL properties on cellulose pretreatment
When neat ILs were used in the pretreatment of cellulose,
At rst, we noticed that Avicel samples regenerated from cellu- those with the “cellulose-dissolving” anions OAcꢂ, Me2PO4
ꢂ
lose-dissolving ILs (i.e., 1, 5, 13, 16–20) displayed much higher and Clꢂ gave the fastest reaction rates (Vmax/Km ¼ 0.21 – 0.46 g
hydrolysis rates (Vmax/Km ranging from 0.21 to 0.46 g Lꢂ1 hꢂ1
)
Lꢂ1 hꢂ1). ILs containing NO3ꢂ, SCNꢂ, Tf2Nꢂ, MeSO4 and Brꢂ
ꢂ
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between the hydrolysis rate and the polarity of neat ILs (see
Fig. 3): a lower IL polarity correlates to a faster hydrolysis. A
likely explanation is that favorable cellulose solvation and
dissolution require balanced van der Waals forces and
hydrophobic interactions,37 which is only partially reected
in the empirical polarity determined by Reichardt's dye.
Conversely, as shown in Fig. S2 and S3,† the Hofmeister
ranking of anions (as characterized by viscosity B-coefficients)
and the hydrophobicity of ILs (as quantied by log P values)
have no direct correlation with the hydrolysis rate of cellulose
pretreated by neat ILs.
Water is known as an “anti-solvent” in cellulose dissolution
because the addition of water rapidly precipitates out cellulose
initially dissolved in an IL. Aqueous solutions of ILs (such as
our 1.0 and 2.0 M solutions studies here) cannot dissolve
cellulose. Consequently, the pretreatment of cellulose by
aqueous ILs is referred to as “suspension pretreatment”. As
shown in Table 1, for a 2.0 M concentration, ILs comprising
the anions MeSO3ꢂ, SCNꢂ, Tf2Nꢂ, BF4ꢂ, Brꢂ and OAcꢂ gave the
fastest hydrolysis with Vmax/Km ranging froꢂm 0.13 to 0.ꢂ16 g Lꢂ1
hꢂ1. ILs based on Clꢂ, Me2PO4ꢂ, MeSO4 and NO3 led to
moderate rate increases (Vmax/Km ¼ 0.11–0.12 g Lꢂꢂ1 hꢂ1) whilst
the remaining ILs (CF3COOꢂ, dcaꢂ, PF6ꢂ, HSO4 and OTfꢂ)
actually showed about the same (or slightly lower) hydrolysis
rate as untreated Avicel.
Fig. 1 Summary of anion effect on the hydrolysis rates of Avicel PH-
101 pretreated by [BMIM]+ type ILs.
anions yielded only moderately improved rates (Vmax/Km ¼ 0.12
– 0.13 g Lꢂ1 hꢂ1) while Avicel treated with ILs containing the
remaining anions (dcaꢂ, CF3COOꢂ, BF4ꢂ, MeSO3ꢂ, PF6 and
ꢂ
OTfꢂ) showed about the same hydrolysis rates as the untreated
cellulose. The high hydrogen-bond basicity of certain anions (as
designated by a high Kamlet–Ta b parameter) has been
credited with breaking up key inter- and intra-molecular
hydrogen-bonds within cellulose, leading to biomass dissolu-
tion in favorable ILs.24,35,36 As illustrated in Fig. 2, a correlation
between the observed Avicel hydrolysis rates and the Kamlet–
Ta b value exists for cellulose treated with neat ILs. In general,
a higher hydrogen-bond basicity coincides with a higher
hydrolysis rate, although exceptions such as OTfꢂ and Tf2Nꢂ are
apparent. In addition, we established a weak correction
When these same ILs are employed at a lower concentration
of 1.0 M, their relative impact on cellulose hydrolysis differs
from the cꢂase of 2.0 M. At 1.0 M, ILs with Clꢂ, MeSO3ꢂ, OAcꢂ,
SCNꢂ, PF6 and Tf2Nꢂ anions resulted in the fastest reactions
(Vmax/Km ¼ 0.13–0.15 g Lꢂ1 hꢂ1), those containing MeSO4
,
ꢂ
NO3ꢂ, BF4ꢂ, Me2PO4ꢂ, dcaꢂ and Brꢂ gave modestly improved
rates (Vmax/Km ¼ 0.11–0.12 g Lꢂ1 hꢂ1), and others (CF3COOꢂ,
HSO4ꢂ,and OTfꢂ) caused no appreciable changes to the reac-
tion rate. In contrast to the use of neat ILs for pretreatment,
aqueous ILs show strikingly different effects on the Avicel
hydrolysis rate and no clear or direct correction between the
Fig. 2 Effect of hydrogen-bond basicities of [BMIM]+-based ILs on the
hydrolysis raꢂte of Aꢂvicel PH-10ꢂ1. [Data refeꢂrences for IL Kamlet–Taft b- Fig. 3 Effect of polarity of [BMIM]+-based ILs on the hydrolysis rate of
values: OAc ,24 Cl ,42 MeSO4 , and SCN ,43 OTfꢂ, BF4ꢂ, Tf2Nꢂ, and Avicel PH-101. [Data reꢂferences forꢂ ETN polarity values: ꢂOAcꢂ,
CF3COOꢂ, and NO3ꢂ
,
Cl ,46 and Tf2N , PF6ꢂ, OTfꢂ, and BF4 47].
ꢂ
PF6 44].
45
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hydrolysis rate and simple physicochemical properties could be (Table 1). Encouragingly, we found that some DESs are as
established, including hydrogen-bond basicity (Fig. 2), polarity effective as aqueous ILs in pretreating cellulose and improving
(Fig. 3), Hofmeister classication (Fig. S2†), or hydrophobicity its subsequent hydrolysis. Further, in aqueous solutions of
(Fig. S3†).
choline chloride (or acetate) mixed with glycerol, we measured
As highlighted in Table 1, pretreatment by several aqueous Vmax/Km values of 0.14 and 0.13 g Lꢂ1 hꢂ1 in 1.0 M solutions of
IL solutions proved effective for improving the hydrolysis rate of 22 and 23, respectively. Remarkably, we also witnessed that a 1.0
cellulose. These solutions included [BMIM]Cl (1.0 M: Vmax/Km
¼
M solution outperforms a 2.0 M solution, the latter frequently
0.15 g Lꢂ1 hꢂ1), [BMIM][OAc] (1.0 M: Vmax/Km ¼ 0.14 g Lꢂ1 hꢂ1), giving no advantage over untreated Avicel. Overall, these results
[BMIM][MeSO3] (1.0 M: Vmax/Km ¼ 0.14 g Lꢂ1 hꢂ1; 2.0 M: Vmax
/
suggest a largely untapped potential for aqueous DESs in the
Km ¼ 0.16 g Lꢂ1 hꢂ1), and [BMIM][SCN] (1.0 and 2.0 M: Vmax/Km pretreatment of cellulose.
¼ 0.14 g Lꢂ1 hꢂ1). An earlier study by Brandt et al.22 suggested
that 80% (v/v) aqueous solutions of [BMIM][HSO4], [BMIM]
3.3 Cellulose structural characterization by XRD, SEM, TGA
and adsorption isotherm
[MeSO3], and [BMIM][MeSO4] were effective for the delignica-
tion and improved digestibility of cellulose. These authors also
indicated that no correlation exists between the pretreatment
effectiveness and the hydrogen-bond basicity of the ILs. In our
hands, neat [BMIM][HSO4] caused considerable cellulose
degradation at 110 ꢀC, resulting in little recovered cellulose. For
this reason, the highest level of [BMIM][HSO4] used was 4.0 M,
as noted in Table 1. In either case, [BMIM][HSO4] proved inef-
fective in generating a rapid cellulose hydrolysis.
To better elucidate how ionic solvents perturb the cellulosic
structure, we performed X-ray diffraction (XRD), scanning
electron microscopy (SEM), and thermogravimetric analysis
(TGA) analysis on untreated Avicel alongside several represen-
tative treated samples. As shown in Fig. 4a, XRD studies indicate
that cellulose regenerated from [BMIM][OAc] has a much lower
crystallinity (35%) than untreated Avicel (79%). In contrast,
It emerges from the entries in Table 1 and Fig. 1 that
pretreatment by several IL systems actually causes a slight
reduction (Vmax/Km below 0.10 g Lꢂ1 hꢂ1) in cellulose hydrolysis.
One probable reason is that residual ILs in IL-treated cellulose
might lead to cellulase inactivation. As shown in Table S1,† the
calculated cellulose recovery may actually exceed 100%, espe-
cially among regenerated celluloses. This is an indication that
IL residues likely become embedded within cellulose chains
and resist efforts to wash it away. In an earlier study, we were
able to both detect residual ILs aer the complete hydrolysis of
cellulose and to conrm that the presence of ILs inactivated the
cellulase.33
The cationic structure of the IL may also moderate the
pretreatment efficiency. As suggested by the entries of Table 1,
the hydrolysis rate of cellulose pretreated by [HMIM][OAc] (17)
is slower than those pretreated by [EMIM][OAc] (16) and [BMIM]-
[OAc] (5). There are two likely reasons for this: (1) rstly, a longer
alkyl chain leads to a larger molar volume, effectively lowering
the anion concentration, which in turn minimizes the disrup-
tive capability of the anion.9 (2) In addition, the residual IL
lingering in the recovered cellulose (see Table S1†) may prompt
dissimilar degrees of cellulase deactivation. Notably, the three
ILs prepared from cations bearing ether functionality (18, 19,
20) are capable of dissolving cellulose and enabling its fast
enzymatic hydrolysis (Vmax/Km ¼ 0.25–0.40 g Lꢂ1 hꢂ1) (Table 1).
These ether-containing ILs developed previously in our
group9,10,38 have the advantages of relatively low viscosity and
low cost, potentially high biodegradability, and minimal cellu-
lase inactivation, the latter due in part to the low anion
concentration.
Cousins to the conventional IL, deep eutectic solvents (DESs,
frequently choline based) are extensions of and alternatives to
ILs and deliver several attractive properties such as low cost, low
toxicity, high biodegradability and high enzyme compatibility.39
For this reason, we examined three DESs (21, 22, 23) and their
Fig. 4 XRD patterns for untreated Avicel PH-101 cellulose and Avicel
aqueous solutions in the pretreatment of Avicel cellulose pretreated by (a) [BMIM][OAc] or (b) [BMIM][MeSO3].
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cellulose samples pretreated by aqueous [BMIM][OAc] (1.0 and
2.0 M) exhibit no such reduction in crystallinity. The changes in
cellulose crystallinity correlate nicely with the enzymatic
hydrolysis rate of cellulose (Table 1). Earlier studies10,40 showed
that cellulosic regeneration from an IL is oen accompanied by
a change in structure from cellulose I to cellulose II. Repre-
sentative SEM images further corroborate that the morphology
of untreated cellulose (Fig. 5a) is more heterogeneous consist-
ing of large particles, while the regenerated cellulose (Fig. 5b)
appears to be more homogeneous and uniform; celluloses
treated by aqueous [BMIM][OAc] (1.0 and 2.0 M) (Fig. 5c and d)
seem to be in-between the heterogeneous and homogeneous
morphologies. In the case of [BMIM][MeSO3], however, Avicel
cellulose pretreatment gave no reduction in crystallinity
(Fig. 4b) for either neat IL or aqueous ILs. From these results, it
should be clear that the cellulose structural changes and
accessibility to cellulase cannot be solely predicted from the
XRD crystallinity index.
Fig. 5 SEM images illustrating the morphology of (a) untreated Avicel
cellulose, (b) Avicel regenerated from [BMIM][OAc], (c) Avicel pre-
treated by 2.0 M [BMIM][OAc], and (d) Avicel pretreated by 1.0 M
[BMIM][OAc].
The alteration in cellulose structure caused by pretreatment
can also be reected in the thermal stability of the resultant
cellulose. Toward this, we conducted TGA experiments on
cellulose samples prior to and following various pretreatment
Table 2 Thermogravimetric results of cellulosic samplesa
Sample pretreatment
Early mass lossb
Tder/ꢀCc
Tdcp/ꢀCd
Transition shapee
Residual charf
Avicel, untreated
5.6%
4.8%
4.7%
4.3%
4.7%
4.1%
4.9%
5.2%
10.9%
8.0%
3.6%
345
280
370
370
323
355
351
332
294
351
346
323
222
300
301
281
325
323
250
232
250
248
S
M
S
S
S
S
S
B
B
B
B
1.1%
24.8%
8.7%
3.0%
6.9%
Avicel, neat [BMIM][OAc]
Avicel, 2.0 M [BMIM][OAc]
Avicel, 1.0 M [BMIM][OAc]
Avicel, neat [BMIM][MeSO3]
Avicel, 2.0 M [BMIM][MeSO3]
Avicel, 1.0 M [BMIM][MeSO3]
Switchgrass, untreated
Switchgrass, neat [BMIM][OAc]
Switchgrass, 2.0 M [BMIM][OAc]
Switchgrass, 1.0 M [BMIM][OAc]
6.6%
4.6%
22.0%
15.4%
5.3%
17.3%g
a
TGA scans were measured on a TA Instruments TGA Q50 under a nitrogen atmosphere using Pt pans with a heating rate of 10 ꢀC minꢂ1. See footer
graphic for a diagram illustrating the measurement of the various transitions and mass loss steps tabulated; * and † indicate the early mass loss step
b
and the position for determination of residual char, respectively. The early mass loss at temperatures below 100 ꢀC is attributed primarily to
c
buoyancy effects and, for this reason, is disregarded in the estimation of Tdcp
.
Tder is determined from the maximum in the rst-derivative
d
envelope of the TGA scan. Tdcp is the decomposition temperature measured at the onset of decomposition (i.e., 10% mass loss following the
initial plateau beyond the early mass loss). The main mass loss behavior is characterized on the basis of whether it occurs as a single, discrete
e
step (S) or exhibits multi-step (M) thermal decomposition. (B) Indicates a derivative thermogram which essentially occurs as a single step but
f
with a slightly bimodal appearance, such as a pronounced shoulder (see Fig. S4–S6†). The amount of carbonaceous char is determined from
g
the mass remaining at 600 ꢀC; uncertainties are estimated to be on the order of ꢄ1–2%. In contrast to the other samples, the residual char
observed for this particular sample showed a large variability.
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strategies. Two representative stability parameters are given in cellulase-binding capability of cellulose, which further implies
Table 2: (i) the decomposition temperature (Tdcp) denotes the the porousness and accessibility of cellulose. As shown in Fig. 6,
thermal onset of decomposition at a 10% total mass loss; and celluloses pretreated by aqueous [BMIM][MeSO3] (1.0 and 2.0
(ii) Tder is the maximum displayed in the rst-derivative of the M) exhibit a higher enzyme-adsorption capacity than that
TGA scan. We note that although Tdcp is most seen in the recent treated by neat [BMIM][MeSO3], suggesting the aqueous IL-
literature, we provide both indices for completeness. Untreated pretreatment leads to a higher accessible surface area and more
cellulose displays a fairly high Tdcp of 323 ꢀC. Typically, cellulose binding sites for cellulase. This explains the higher hydrolysis
10
pretreatment results in structural changes which reduce Tdcp
.
rates of celluloses pretreated by aqueous [BMIM][MeSO3] than
Thus, to an extent, the Tdcp value reects the severity of struc- that by neat IL (Table 1). Fig. S7† indicates no signicant
tural changes occurring as a result of pretreatment. As indicated adsorption differences among cellulose samples pretreated by
in Table 2, regenerated cellulose from [BMIM][OAc] has a much neat or aqueous choline chloride/glycerol (1 : 2). Although the
lower Tdcp (222 ꢀC) than cellulose pretreated by aqueous exact mechanism of how aqueous ILs interact with cellulose
ꢀ
[BMIM][OAc] solution (ꢁ300 C). This accords with our earlier chains is not clear, it is helpful to offer some insightful
XRD and SEM results, as well as the observed hydrolysis rates. discussions. When neat or aqueous ILs are not capable of dis-
Namely, the process of dissolving cellulose in neat [BMIM][OAc] solving cellulose, these solvent molecules may not considerably
signicantly modies the cellulose crystallinity and makes the disrupt the hydrogen-bonding network to make noticeable
cellulose more susceptible to hydrolysis whereas aqueous structural changes as detected by XRD and TGA. However, in
[BMIM][OAc] has minimal impact on the cellulose structure. aqueous ILs, hydrated ions may have better penetration/diffu-
However, this analysis appears less applicable in the case of sion power than (non-cellulose dissolving) neat IL molecules to
[BMIM][MeSO3] pretreatment. Specically, cellulose pretreated weakly disrupt the hydrogen-bonds, enable subtle morphology
by neat [BMIM][MeSO3] shows a signicant reduction in changes, and increase the cellulose accessibility and
thermal stability (Tdcp ¼ 281 ꢀC), however, Vmax/Km is only 0.093 digestibility.
g Lꢂ1 hꢂ1 in this case. Contrariwise, cellulose samples treated by
aqueous [BMIM][MeSO3] show essentially an identical Tdcp as
untreated Avicel (Table 2 and Fig. S5†). However, cellulose
hydrolysis kinetics analysis clearly reveals a favorable boost
3.4 IL pretreatment of switchgrass
Switchgrass (Panicum virgatum L.) is known to be a viable source
of lignocellulose for bioethanol production.41 Given its rele-
vance to bioenergy, we initiated studies pretreating switchgrass
with [BMIM][OAc], [BMIM][MeSO3], and their aqueous solu-
tions (Fig. 7 and 8). As shown in Fig. 7 and 8, pretreatment by
both neat [BMIM][OAc] (which partially dissolves the grass) and
[BMIM][MeSO3] signicantly sped up the enzymatic hydrolysis
of cellulose. In addition, pretreatment with neat [BMIM][OAc]
improved the hydrolysis of hemicellulose. Aqueous [BMIM]-
[OAc] solutions proved to be much less effective for pretreating
switchgrass and gave low hydrolysis rates for both cellulose and
hemicellulose. XRD patterns (Fig. 9a) conrm a substantially
upon pretreatment with aqueous [BMIM][MeSO3] (1.0 M: Vmax
/
Km ¼ 0.14 g Lꢂ1 hꢂ1; 2.0 M: Vmax/Km ¼ 0.16 g Lꢂ1 hꢂ1). From
this, we can conclude that XRD and TGA studies may not be able
to reveal some subtle but important structural changes that
occur during cellulose pretreatment with ILs and their aqueous
solutions.
To probe such subtle structural changes, we carried out the
cellulase adsorption isotherm of Avicel celluloses pretreated by
various ionic solutions (Fig. 6 and S7†). The adsorption exper-
iments were carried out at 4 ꢀC to minimize the enzymatic
hydrolysis of cellulose. The adsorption isotherm measures the
Fig. 6 T. reesei cellulase adsorption isotherm (4 ꢀC) of Avicel PH-101
pretreated by neat or aqueous [BMIM][MeSO3].
Fig. 7 Enzymatic hydrolysis of switchgrass pretreated by [BMIM][OAc].
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Fig. 8 Enzymatic hydrolysis of switchgrass pretreated by [BMIM]
[MeSO3].
Fig. 10 SEM images illustrating the morphology of (a) untreated
switchgrass, (b) switchgrass regenerated from [BMIM][OAc], (c)
switchgrass pretreated by 2.0 M [BMIM][OAc], and (d) switchgrass
pretreated by 1.0 M [BMIM][OAc].
reduced crystallinity (23%) for switchgrass regenerated from
neat [BMIM][OAc] (cf. 41% for untreated grass), and a lower, but
still substantial, reduction in crystallinity following aqueous
[BMIM][OAc] treatment (1.0 M: 27%; 2.0 M: 30%). The associ-
ated SEM images shown in Fig. 10 indicate that switchgrass
samples pretreated by aqueous [BMIM][OAc] retain a hetero-
geneous morphology similar to the untreated grass, while the
grass regenerated from neat [BMIM][OAc] appears to be more
homogeneous.
Alternatively, aqueous [BMIM][MeSO3] solutions proved
surprisingly effective as pretreatment agents to produce high
hydrolysis rates for both cellulose and hemicellulose. These
observations are in agree with our above results demonstrating
that aqueous [BMIM][MeSO3] is a promising treatment medium
for processing Avicel cellulose. However, as revealed in Fig. 9b,
there is no perceptible reduction in crystallinity observed for
switchgrass samples pretreated by either neat [BMIM][MeSO3]
or aqueous [BMIM][MeSO3]. This observation supports the
notion that, in spite of no evident change in crystallinity, certain
pretreatments can lead to subtle changes that markedly impact
the cellulose accessibility and digestibility.
4 Conclusions
In this research, we have systematically studied the hydrolysis
rates of cellulose and switchgrass pretreated by neat ILs, DESs,
and their aqueous solutions. The hydrolysis rate of cellulose
treated by neat ILs can be correlated with the H-bond basicity of
the constituent anions and the empirical IL polarity, however,
the hydrolysis rates of cellulose pretreated by aqueous ILs
cannot be simply related to any single property of ILs (e.g., H-
bond basicity, polarity, Hofmeister ranking, and hydropho-
bicity). We further examined the structural changes of cellulosic
biomass upon pretreatment by IL solutions using XRD, SEM,
Fig. 9 XRD patterns for untreated switchgrass and switchgrass pre-
treated by (a) [BMIM][OAc] and (b) [BMIM][MeSO3].
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TGA and adsorption isotherm characterizations. In the case of 14 Q. Zhang, N. S. Oztekin, J. Barrault, K. De Oliveira Vigier and
´ ˆ
[BMIM][OAc], the hydrolysis rates for Avicel and switchgrass
F. Jerome, ChemSusChem, 2013, 6, 593–596.
correspond to observable structural changes: particularly, a 15 Q. Zhang, M. Benoit, K. De Oliveira Vigier, J. Barrault and
´ ˆ
reduction in crystallinity, a homogeneous morphology, and a
F. Jerome, Chem.–Eur. J., 2012, 18, 1043–1046.
reduced thermal stability. However, in the case of [BMIM]- 16 R. Rinaldi, Chem. Commun., 2011, 47, 511–513.
[MeSO3], the hydrolysis rates of (ligno)cellulose can be related to 17 M. Francisco, A. van den Bruinhorst and M. C. Kroon, Green
neither the crystallinity nor the thermal stability of the cellu-
lose; but a higher cellulase-adsorption trend was observed for 18 M. Gericke, T. Liebert, O. A. El Seoud and T. Heinze,
celluloses pretreated by aqueous IL solutions than that by neat Macromol. Mater. Eng., 2011, 296, 483–493.
IL. Clearly, multifaceted or elusive structural changes must be 19 X. Tian, Z. Fang, D. Jiang and X. Sun, Biotechnol. Biofuels,
implicated in this case. From this work, aqueous solutions of 2011, 4, 53.
several ILs and DESs emerge as effective pretreatment agents for 20 D. Fu and G. Mazza, Bioresour. Technol., 2011, 102, 7008–
cellulose, including [BMIM][MeSO3] (7), [CH3(OCH2CH2)3–Et- 7011.
Pip][OAc] (20), [BMIM][OAc] (5), [EMIM][OAc] (16), [BMIM]Cl 21 D. Fu and G. Mazza, Bioresour. Technol., 2011, 102, 8003–
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