Production and extraction of sugars from switchgrass hydrolyzed in ionic liquids
© Sun et al.; licensee BioMed Central Ltd. 2013
Received: 2 November 2012
Accepted: 8 March 2013
Published: 20 March 2013
The use of Ionic liquids (ILs) as biomass solvents is considered to be an attractive alternative for the pretreatment of lignocellulosic biomass. Acid catalysts have been used previously to hydrolyze polysaccharides into fermentable sugars during IL pretreatment. This could potentially provide a means of liberating fermentable sugars from biomass without the use of costly enzymes. However, the separation of the sugars from the aqueous IL and recovery of IL is challenging and imperative to make this process viable.
Aqueous alkaline solutions are used to induce the formation of a biphasic system to recover sugars produced from the acid catalyzed hydrolysis of switchgrass in imidazolium-based ILs. The amount of sugar produced from this process was proportional to the extent of biomass solubilized. Pretreatment at high temperatures (e.g., 160°C, 1.5 h) was more effective in producing glucose. Sugar extraction into the alkali phase was dependent on both the amount of sugar produced by acidolysis and the alkali concentration in the aqueous extractant phase. Maximum yields of 53% glucose and 88% xylose are recovered in the alkali phase, based on the amounts present in the initial biomass. The partition coefficients of glucose and xylose between the IL and alkali phases can be accurately predicted using molecular dynamics simulations.
This biphasic system may enable the facile recycling of IL and rapid recovery of the sugars, and provides an alternative route to the production of monomeric sugars from biomass that eliminates the need for enzymatic saccharification and also reduces the amount of water required.
Lignocellulosic biomass is a renewable resource that may be converted to fuels and/or chemicals [1, 2]. Recent research and development efforts have examined a two step bioconversion process that involves: 1) liberation of fermentable sugars from lignocellulose and, 2) conversion of sugars into fuels and/or chemicals by fermentation [3, 4]. The potential of lignocellulosic biomass to serve as a renewable feedstock has not been realized, primarily due to the historical shift towards petroleum-based feedstocks in the 1920s and the difficulty in depolymerization of lignocellulose into its component monomeric sugars .
The use of ionic liquids (ILs) as biomass solvents is an attractive alternative for the pretreatment of lignocellulosic biomass . It has been shown that pretreatment with imidazolium-based ILs containing anions such as chloride , acetate  and alkyl phosphate , can greatly accelerate the subsequent enzymatic hydrolysis of biomass. Current approaches that use neat IL as the pretreatment solvent and water as antisolvent to precipitate carbohydrate-rich material require significant amounts of water to extract residual IL from the precipitated cellulose and undissolved biomass, and also require an effective means to recover and recycle the IL [8, 10]. These requirements pose significant economic and sustainability challenges to the deployment of the IL pretreatment technology .
Another approach to sugar production using ILs is to use acid catalysts to produce sugars and other compounds in situ through the hydrolysis of polysaccharides dissolved in imidazolium chloride [12–15]. Li et al. reported biomass hydrolysis in ILs with different mineral acids as catalysts and achieved a maximum 81% liberation of the total reducing sugars initially present in the biomass with 1-n-butyl-3-methylimidazolium chloride ([C4mim]Cl) and hydrochloric acid . The use of Brønsted acid ILs, which act as both the solvent and catalyst, to dissolve and hydrolyze cellulose has also been reported . This could potentially provide a means of liberating fermentable sugars from biomass without employing enzymatic saccharification [11, 17]. Separation of the sugars from the aqueous IL and recovery of the IL after acid hydrolysis are challenging and must be addressed in order to make this process economically viable.
Rogers et al. reported that certain hydrophilic ILs could form an aqueous biphasic system (ABS) in the presence of concentrated kosmotropic salts . Subsequently, significant progress has been made that demonstrates the efficacy of this approach for separation of biomolecules, small organic molecules, biochemicals, and radiological isotopes [19–23]. It has been reported that an ABS can be formed with addition of an appropriate amount of K3PO4, K2HPO4, K2CO3, KOH, NaOH, or Na2HPO4 to an aqueous solution containing [C4mim]Cl [19, 24]. When added to certain aqueous IL solutions, kosmotropic anions stabilize water-water interactions, resulting in more energy being required for cavity formation around the bulky organic [C4mim]+ cation. At a certain concentration of kosmotropic salts, an aqueous phase containing chaotropic IL can phase separate with the salt phase . We describe a process that uses the phase separation behavior of imidazolium ILs/alkali/water solutions in tandem with acid catalyzed hydrolysis to extract the sugars liberated from biomass from the aqueous IL solutions. This approach offers the potential of reducing costs of sugar production from lignocellulose by eliminating the need for enzymes and decreasing the water consumption requirements of more traditional IL pretreatment approaches.
Results and discussion
Alkali extraction using sugar standards
Sugar concentrations before addition of NaOH correspond to 90–100% of the sugars added to the aqueous [C2mim]Cl or [C4mim]Cl solution, indicating minimal degradation of sugars under the conditions used for acidolysis. It was observed that more glucose is extracted to the bottom phase in comparison to xylose. For the upper IL rich phase, less than 1% glucose or xylose can be detected. The chromatograms of the upper and lower phases are shown in Additional file 1: Figure S1. It should be noted that due to the significantly different sugar concentrations present, the lower phase aliquot was diluted 3000× in order to be quantified by High Performance Anion Exchange Chromatography (HPAEC); however, the upper phase was only diluted 5×. The system using [C4mim]Cl was found to be more efficient, with better extractions for both glucose and xylose ([C4mim]Cl with 15% NaOH: 96.5% for glucose, 73.9% for xylose vs. [C2mim]Cl with 15% NaOH: 90.1% for glucose, 59.2% for xylose). With higher concentrations of NaOH, the amount of glucose partitioned to the lower phase is slightly higher ([C4mim]Cl: 96.5% with 15% vs. 98.3% with 20%; [C2mim]Cl: 90.1% with 15% vs. 92.0% with 20%), while the amount of xylose in the bottom phase decreased ([C4mim]Cl: 73.9% with 15% vs. 60.9% with 20%; [C2mim]Cl: 59.2% with 15% vs. 56.7% with 20%). We hypothesized that this was due to the degradation of the xylose in strongly basic conditions. Based on the results obtained with sugar standards, [C4mim]Cl/15% NaOH system was selected as the system for subsequent experiments with switchgrass.
The total potential energies of the simulated systems
Sugar + system
ΔΔE (per glucose)
The calculated interaction energy of glucose with the NaOH/water systems is more preferred (lower interaction energy) than the interaction energy of glucose in the IL/water system, indicating that glucose tends to partition into the alkaline phase. The more preferred interactions between glucose and NaOH/water solutions also indicates that the hydroxyl group forms stronger hydrogen bonds with the anionic OH- group due to the stronger charge distribution around the OH- anions. The calculated partition coefficient is 2.91 and agrees well with the experimental result of 3.18. These results suggest that MD simulations can be used to understand the partitioning of monomeric sugars into various IL/water and NaOH/water systems and to predict partition coefficients of sugars into these systems to assist in the choice of the IL.
Biomass pretreatment and acidolysis
Glucose and xylose yields after acidolysis of biomass in [C 4 mim]Cl using different pretreatment conditions and water addition methods a
H2O addition method
Solid residue (wt%)
Glucose yield (%)
Xylose yield (%)
105°C 6 h
20.7 ± 0.4
99.8 ± 2.6
105°C 6 h
24.2 ± 0.3
98.6 ± 1.8
105°C 6 h
27.4 ± 0.7
95.1 ± 2.0
160°C 1 h
37.1 ± 1.2
83.4 ± 3.8
160°C 1.5 h
69.4 ± 2.5
81. 9 ± 2.8
160°C 1.5 h
Pumped @ 10 min
38.6 ± 2.7
92.6 ± 5.5
160°C 1.5 h
Pumped @ 15 min
83.3 ± 1.9
52.1 ± 1.2
In previous reports , water was added at defined time intervals (0 min, 10 min, 20 min, 30 min and 60 min) during acidolysis to achieve higher sugar yields. As this may not be a practical approach to water addition, we evaluated the impact of pumping water into the system using a syringe pump, and the results are compared from runs 5 to 7 Table 2. Runs 6 and 7 indicate that the time interval when the water was pumped into the system has a significant impact on the observed sugar yield. For example, in run 6, water was pumped in 10 min after the initial addition of water and acid; the glucose yield was observed to decrease significantly (Run 5 69.4% vs. Run 6 38.6%) although they were pretreated under the same conditions: 160°C for 1.5 h. When the reaction interval was changed to 15 min with 20% water addition performed at 10 min, the glucose yield improved from 69.4% to 83.3%. We hypothesize that this result can be attributed to the kinetics of the glucose hydrolysis based on observations made in previous reports , and the optimized water addition are plotted in Additional file 1: Figure S2. Figure 3 shows clearly that the maximum glucose yields occur at the expense of reduced xylose yields, which is expected, as xylan is easier to hydrolyze compared to glucan. At more severe process conditions, more glucan can be broken down but this also results in spontaneous xylose degradation.
Glucose and xylose yields after acidolysis of biomass in [C 4 mim]Cl using different solids loadings a
Solid residue (wt%)
Glucose yield (%)
Xylose yield (%)
Final [Glc] (g/L)
Final [Xyl] (g/L )
83.3 ± 1.9
52.1 ± 1.2
8.0 ± 0.2
3.4 ± 0.1
55.5 ± 3.0
34.1 ± 2.0
8.3 ± 0.4
3.5 ± 0.2
54.4 ± 1.6
32.9 ± 2.8
10.2 ± 0.3
6.7 ± 0.4
56.2 ± 3.1
36.7 ± 1.9
15.5 ± 0.8
6.9 ± 0.4
Sugar extraction after acidolysis of biomass
Partition of the sugars after phase separation
8.55 ± 0.65
18.58 ± 1.50
2.31 ± 0.13
7.47 ± 0.41
2.46 ± 0.11
3.62 ± 0.24
8.84 ± 0.13
5.71 ± 0.10
17.84 ± 0.27
8.24 ± 0.05
10.07 ± 0.17
8.10 ± 0.17
24.73 ± 1.39
7.00 ± 0.65
26.56 ± 0.28
15.20 ± 0.49
28.06 ± 0.26
26.91 ± 0.42
32.94 ± 2.86
30.86 ± 3.19
Quantification of the HMF and furfural in the system
% Glu. to HMF
% Xyl. to furfural
2.16 ± 0.00
6.07 ± 0.01
1.79 ± 0.01
6.05 ± 0.02
2.05 ± 0.01
5.63 ± 0.02
2.95 ± 0.01
5.88 ± 0.01
4.79 ± 0.06
8.52 ± 0.02
3.25 ± 0.03
9.01 ± 0.02
5.14 ± 0.02
10.93 ± 0.00
3.66 ± 0.02
4.66 ± 0.04
3.20 ± 0.00
6.23 ± 0.01
4.53 ± 0.00
9.35 ± 0.07
Quantification of the IL in the lower alkaline rich phase
[C4mim]Cl in alkali phase (mM)
% of IL to the alkaline phase
Mass balance and characterization of the solid residue
In contrast, the biomass pretreatment at higher temperature results in disappearance of the broad peak at ca. 15–16º, which represents a combination of the 101 and 10ī planes of cellulose I. The material is highly amorphous with a minor crystalline component (CrI = 0.08). The broad peak around 21.4° may be assigned to the 002 cellulose II lattice plane. This indicates that the solvent IL has penetrated inside the solid part and disrupted the crystal structure of cellulose during the higher temperature pretreatment. This may explain why higher temperature/shorter time pretreatment is more efficient in solubilizing the biomass, thus resulting in more sugar production. Another possible explanation for the observed structural change is that the relative ratio of the three major biomass components is altered as a result of the pretreatment.
Lignin molecular weight distribution (calculated from SEC data in Additional file 1 : Figure S5)
Elution time (min)
t < 15
t > 15
Molecular mass (u)*
46 k < u
46 k > u
Before phase separation
It has been shown that certain concentrations of NaOH can phase separate with chloride based ILs ([C4mim]Cl and [C2mim]Cl) forming upper phase, IL rich and lower phase, alkaline rich. Both glucose and xylose prefer to partition to the alkaline rich phase. By combining this system with the acidolysis of biomass in IL, sugar monomers can be easily extracted from the aqueous ILs. The sugar yields depend on both the pretreatment conditions and alkali concentrations. Pretreatment under higher temperature results in improved glucose yields but compromises xylose yields. The optimized NaOH concentration for both phase separation and sugar extraction is 15%. Maximum yields of 54% glucose and 88% xylose can be recovered in the alkaline phase with pretreatment condition of 160°C for 1.5 h and 105°C for 6 h respectively followed by acidolysis. Improved sugar yields could be achieved by further optimizing the amount of acid and water used in acidolysis step and the alkali salts used for sugar extraction. Molecular dynamics simulations can be used to predict sugar partitioning in the system, but the sugar partition coefficients are found to be affected by the presence of biomass.
Consolidated pretreatment and hydrolysis using ILs and acid catalysts offers a promising route to the production of fermentable sugars without the need for enzymes. The use of alkali salts to form ABS to recover the sugars and recycle the IL may provide a scalable and economical process. The advantages of the process are: 1) sugars can be released in situ and extracted by alkaline solution with relatively high yields, and without the need for any enzymes; 2) the formation of an aqueous biphasic system enables facile recovery of the sugars and IL recovery at the same time; 3) significantly reduced volume of water (< 50 wt% of total mixture) is used as compared to more traditional IL based pretreatment process. Future research should be focused on recovery of the residual biomass in the IL rich phase, test the IL recycling efficiency, and desalting of the alkaline rich phase to make it compatible with downstream fermentation microbes.
Biomass pretreatment and acidolysis of dissolved biomass
The pretreatment and acidolysis process flow is shown in Additional file 1: Figure S6. Biomass solutions were prepared by combining different amounts (0.5 g, 0.75 g, 1 g, and 1.5 g) of switchgrass with 10 g [C4mim]Cl in an 80 mL glass bottle. The mixtures were heated and stirred in an oil bath at different conditions. All experiments were conducted in duplicates. Solutions were then placed into another oil bath which was already equilibrated at the acidolysis temperature of 105°C and acidolysis started after 15 min equilibration.
Acidolysis was performed following a procedure described previously . In summary, 4 M HCl was added to the biomass-[C4mim]Cl solution (t = 0) at concentrations of 100 mg HCl per g biomass and with DI water added to give a H2O concentration of 5% (w/w) of the total weight. More water was added at different time intervals (10 min, 20 min, 30 min, and 60 min) to result in targeted water concentrations of 20, 25, 33 and 43%. Continuous water addition using a syringe pump was also attempted to compare the effect on sugar yields. Water was pumped into the mixture starting from either 10 or 15 min at the rate of 157.2 or 121.1 uL/min for 50 or 45 min. Acidolysis was continued for a total of 2.5 h and stopped by taking the bottle out of the oil bath with/without addition of extra amount of water (0, 7.5, or 15 mL). The mixture was transferred into centrifuge tubes and centrifuged at high speed (10,000 rpm) to separate the solid residue from the aqueous solutions. The solid residue was washed with 5× 40 mL of water, and after the final wash the sample was lyophilized for two days for further analysis.
Extraction of sugars using alkaline solution
Extraction of sugar standards
33 mg glucose and 21 mg xylose were used to simulate the sugar outputs obtained from 0.1 g biomass. These sugars were dissolved in an IL-H2O mixture (2 g IL + 1.5 g H2O) in a 15 mL centrifuge tube. 70 μL of 4 M HCl was added to the mixture and mixed in an incubator at 30°C and 1400 rpm for 30 min. 1 mL of the mixture solution was placed in to a 2 mL eppendorf tube and different amounts (ca. 130 or 200 uL) of concentrated NaOH (50% w/w) were added to give the final NaOH concentration either 15 or 20 wt% (considering the water in the system). The mixture was agitated in a thermomixer at RT and 1400 rpm for 0.5 h and then centrifuged at high speed (14,000 rpm) to phase separate. The upper IL phase and lower NaOH phase were separated with a pipette and the sugar content was quantified. The volume of the upper and lower phase was calculated by measuring the mass and density of both phases.
Extraction of acidolysis sugars
The procedure is similar to the extraction of sugar standard except that only 15 wt% NaOH (final concentration, based on the water in the system) was used based on the results from the sugar standards. The total volume of the supernatant was calculated based on the total mass and density of the supernatant after separation of the solid residue.
Analysis and characterization methods
All aqueous solutions were analyzed for sugars using High Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) on a Dionex ICS 3000 equipped with a Dionex CarboPac PA-20 analytical column (3 × 150 mm), according to procedures described previously [8, 33]. Elution was initiated with 89% (v/v) water and 11% (v/v) 1 M NaOH for the first 13.5 min, with 10 μL injection volume and 0.4 mL/min for the flow rate. A 5 min gradient was applied and elute concentration was then switched to 55% (v/v) water and 45% (v/v) 100 mM NaOH for 30 min. Sugar standards fucose, arabinose, rhamnose, galactose, glucose, xylose, fructose, and cellubiose obtained from Sigma-Aldrich were used as the external standards for HPAEC, and prepared at levels of 6.25 to 100 μM before use.
Furfural and HMF was analyzed using an Agilent 1200 High Pressure Liquid Chromatography (HPLC) instrument equipped with Aminex HPX-87 H column and a UV detector ( λ = 280 nm). Eluent containing 4 mM H2SO4 was used and the flow rate was 0.6 mL/min. Standard calibration curves were made by using 6 different known concentrations of furfural/HMF (125–1000 uM) from Sigma-Aldrich. Ionic liquid was quantified using reversed phase liquid chromatography using an HPLC equipped with Eclipse Plus C8 column and Evaporative Light Scattering Detector (ELSD, evaporator temperature = 45°C, nebulizer temperature = 30°C; gas flow = 1.2). All analyses were performed at 0.5 mL/min flow rate. The injection volume was 5 μL and the column temperature was 30°C.
The chemical composition of the biomass before and after pretreatment was tracked using an acidolysis protocol that followed the NREL Laboratory Analytical Protocols (LAP) LAP-002 and LAP-005 scaled down to the volumes of the samples . In short, 0.2 g biomass and 2 mL 72% H2SO4 was incubated at 30°C with shaking rate of 300 rpm for 1 h. The solution was diluted down to 4% H2SO4 and autoclaved for 1 h at 121°C. The reaction was quenched by placing the samples into an ice bath and then filtered. Carbohydrate concentrations were determined using HPAEC and the acid insoluble lignin was quantified gravimetrically.
NMR samples were ball milled using a Retsch PM 100 planetary ball mill at 600 rpm with a stainless steel grinding jar (50 mL) containing zirconium dioxide balls (10 mm × 10). The samples were milled for 6 h with 5 min grinding intervals with 5 min breaks. 25 mg of these milled biomass samples were mixed with 500 uL pre-mixed DMSO-d6/pyridine-d5 (4:1 v/v) directly in NMR tubes according to the report by Ralph et al. The tubes were placed into an ultrasonic bath with the temperature at 50°C and for 8 h to swell/dissolve the biomass. Heteronuclear single quantum coherence (HSQC) spectra were recorded at 310 K using a Bruker Avance-600 MHz equipped with a cryo-probe (hsqcetgpsisp.2, ns = 128, ds = 16, d1 = 0.5 s, td = 1 k, number of increments = 512). DMSO was used as an internal reference. Topspin was used for processing and analysis of the data.
Size exclusion chromatography (SEC) was employed to assess changes in lignin mass distribution. An EMAL lignin of switchgrass was employed as a control . EMAL has shown to be more representative of the total lignin present in biomass compared to the lignin extracted using other protocols such as milled wood lignin (MWL) or cellulolytic enzyme lignin (CEL) [36, 37]. Lignin solutions were prepared in analytical grade N-methyl-2-pyrrolidinone (NMP) and dimethylsulfoxide (DMSO) (1:1, v/v) with sonication for 3 hours at 40°C. Polydispersity of dissolved lignin was determined using analytical techniques SEC UV-A as previously described . An Agilent 1200 series binary LC system (G1312B) equipped with a DAD (G1315D) was used. Separation was achieved with a Mixed-D column (5 mm particle size, 300 mm × 7.5 mm i.d., linear molecular weight range of 200 to 400,000 u, Polymer Laboratories) at 80°C using a mobile phase of NMP at a flow rate of 0.5 ml min-1. Absorbance of material eluting from the column was detected at λ = 300 nm (UV-A). Molecular mass estimates were determined after calibration of the system with polystyrene standards.
Molecular dynamics simulation of glucose in IL-water-NaOH system
The component of simulated systems
Number of NaOH
Number of water
Number of [C2mim]Cl
Number of glucose
NaOH phase with sugar
IL Phase with sugar
The authors thank Prof. Harvey W. Blanch for his thoughtful and valuable comments and review of this manuscript. This research used resources of the National Energy Research Scientific Computing Center, which is supported by the Office of Science of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231. The work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the U.S. Department of Energy under Contract No. DE-AC02-05CH11231.
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