Nanoscale Observation of Microfibril Swelling and Dissolution in Ionic

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Nanoscale observation of microfibrils swelling and dissolution in ionic liquids Junli Xu, Baocai Zhang, Xingmei Lu, Yihua Zhou, Jinyun Fang, Yao Li, and Suojiang Zhang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.7b03269 • Publication Date (Web): 04 Nov 2017 Downloaded from http://pubs.acs.org on November 5, 2017

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ACS Sustainable Chemistry & Engineering

Nanoscale observation of microfibrils swelling and dissolution in ionic liquids Junli Xu,a Baocai Zhang,b Xingmei Lu,a, c Yihua Zhou,b Jinyun Fang,d Yao Li,a and Suojiang Zhanga, c,* a

Beijing Key Laboratory of Ionic Liquids Clean Process, Key Laboratory of Green Process and

Engineering, State Key Laboratory of Multiphase Complex Systems, Institute of Process Engineering, Chinese Academy of Sciences, 1 North 2nd Street, Zhongguancun, Haidian District, Beijing, PR China, 100190 b

State Key Laboratory of Plant Genomics, Institute of Genetics and Developmental Biology,

Chinese Academy of Sciences, No. 1 West Beichen Road, Chaoyang District, Beijing, P.R. China, 100101 c

School of Chemistry and Chemical Engineering, University of Chinese Academy of Sciences,

19A Yuquan Road, Shijingshan District, Beijing, P.R.China 100049 d

Technical Institute of Physics and Chemistry, Chinese Academy of Sciences, 29 Zhongguancun

East Road, Haidian District, Beijing, P.R.China, 100190

*Correspondence should be addressed to S. J. Zhang (e-mail:[email protected]), Fax: +8601082544875, Tel: +8601082544875

ACS Paragon Plus Environment

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ABSTRACT: Ionic liquids (ILs) are widely used for biomass pretreatment due to their excellent dissolving capacity. The efficient and economical use of ILs in pretreatment greatly depends on revealing the underlying mechanism of biomass dissolution. Here, we observed that cellulose microfibrils of rice straw were swelled in ILs using atomic force microscopy, and the swelling resulted in cellulose crystallinity decrease by time-course X-ray diffraction analysis. The ILinduced cellulose microfibrils swelling greatly promoted the saccharification efficiency of rice straw. Compared with conventional acid or alkali solutions, strong hydrogen bond interactions between IL with appropriate sizes and polysaccharides resulted in cellulose microfibrils swelling in ILs, which was demonstrated by nuclear magnetic resonance and molecular simulation. Therefore, microfibrils swelling is the key step in cellulose dissolution with ILs, effective swelling could accelerate cellulose dissolution and biomass pretreatment. Thus, those results are helpful for understanding mechanism of lignocellulose dissolution in ILs and developing new ILs for biomass pretreatment.

KEYWORDS: Ionic liquids, Biomass, Swelling, Cellulose microfibrils

INTRODUCTION Pretreatment is an essential process in biomass utilization, and a variety of different methods have been developed in recent years.1 Ionic liquids (ILs) has emerged as a promising solvent due to its excellent performance in biomass pretreatment. The IL 1-butyl-3methylimidazolium chloride ([Bmim]Cl) was initially introduced as a reagent for dissolving


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cellulose.2 Currently, ILs composed of varied cations and anions have demonstrated high efficiency in cellulose extraction and delignification.3-4 IL pretreatment has shown unique advantages over traditional pretreatments methods in increasing surface area, reducing cellulose crystallinity and decreasing lignin content,5,6 meanwhile, IL pretreatment methods greatly improved the enzymatic saccharification.7-9 In addition, IL-containing systems were also studied to improve enzymatic reaction10 or cellulose hydrolysis into monomeric glucose.11 Despite many investigations on biomass pretreatment with IL, little agreement exists on the underlying mechanism of lignocellulose dissolution with ILs. The mechanism of cellulose dissolution with ILs is thought to be due to new hydrogen bonds formation between glucan chain and ILs.12,13 Molecular dynamics simulations of cellulose solvated in [Bmim]Cl indicated that the concerted action of anions and cations on the cellulose surfaces.14 All-atom molecular dynamics simulations of small bundles disruption of cellulose Iα and Iβ in different ILs indicated that IL anions strongly bound to the hydroxyl groups of the bundles, weakened the intra-strand hydrogen bonds and IL cations intercalated within individual strands.15 The hydrogen bonds of ILs (ILHBs) are different from the traditional Hydrogen Bonds (HBs), the hydrogen bonds of ILs contain not only the hydrogen bonds interaction between cations and anions, but also the electrostatic interaction induced by the cations and anions.16,

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When cellulose dissolution

occurred in ILs, the electrostatic interaction of cations and anions of ILs could induce the charge deviation of glucan chain hydroxyl, so the special hydrogen bonds containing electrostatic force are formed between ILs and glucan chain. The interaction energy between Bmim+, Cl- and glucan chain are around -708.618 kJ/mol and -1228.34 kJ/mol, respectively.18 Those special hydrogen bonds interactions between ILs and cellulose are similar to the IL-HBs, and those


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characteristics induced the cellulose to dissolve into ILs, these hydrogen bonds between ILs and glucan chain was named as IL-HBs in this work. Meanwhile, nuclear magnetic resonance (NMR) spectroscopy studies indicated that cellulose dissolution in ILs was governed by the interactions between the IL anions and the carbohydrates,19 and hydrogen bonds are formed between cellobiose hydroxyl groups and both anions and cations of 1-ethyl-3-methylimidazolium acetate ([Emim][OAc]).20

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Cl-

NMR relaxation studies have also indicated that the hydrogen bonds were formed between carbohydrate hydroxyl protons and the IL chloride anions during the dissolution of cellulose in ILs.21 In addition to these simulation and NMR studies on the interactions between ILs and glucose/cellobiose, microscopy has been applied to observe the dissolution process in the biomass pretreatment;22 confocal raman microscopy and confocal fluorescence microscopy were used to investigate the process of plant cell wall dissolution by [Emim][OAc] in micron level.2325

However, most descriptions were limited to the general morphological alterations of the plant

cell walls. Alterations of the cellulose microfibrils by IL treatments have not been described in higher-resolution, although atomic force microscopy (AFM) has been applied to image the native microfibrils in plants.26 Despite significant research progress in this field, the IL pretreatment effects on cellulose/biomass, and especially on microfibrils, remains elusive. To elucidate process of plant cell wall dissolution by ILs, we visualized the effects of IL on rice (Oryza sativa L.) cellulose microfibrils by using AFM. Meanwhile, we also used different solvents to treat cellulose microfibrils to compare with the most effective ILs. X-ray diffraction (XRD), NMR, and molecular dynamics (MD) analyses were also introduced into this work to


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analyze cellulose microfibrils morphology and structure changes in ILs and other solvents, released glucose from the IL treated rice straw was used to evaluate the effect of ionic liquids on cellulose microfibers swelling. EXPERIMENTAL SECTION Sample preparation. The wild-type (WT) and brittle culm1 (bc1) rice plants used in this study were cultivated in the experimental fields at the Institute of Genetics and Developmental Biology in Beijing (China) during the natural growing seasons. For AFM microscopy, transverse sections were hand-cut from fresh rice straws using a single-blade razor. The sections were delignified by treatment with 10% NaClO2 at 1% (w/v) of biomass (acidified by 0.1 M HCl) overnight at room temperature.27 For NMR characterization, cellobiose, cellohexaose and were purchased from Sigma-Aldrich (St. Louis, MO, USA). Microcrystalline cellulose (MCC) was purchased from Sinopharm Chemical Reagent Co., Ltd. The acid-treated cellulose was obtained from a mixed acid solution (H2O:HNO3:HOAc = 2:1:8 (v:v:v)).28 Ten milligrams of WT rice straw powder was treated with 1 mL of the mixed acid solution for 30 min at 100°C. After centrifuging the solution at 12000 rpm for 5 min, the pretreated rice straw was washed with deionized water until reaching a neutral pH, followed by drying for NMR characterization. AFM and XRD characterization. For atomic force microscopy, the delignified slices of rice straw were imaged with a MultiMode scanning probe microscope (MM-SPM) with an advanced NanoScope V Controller (Veeco, Santa Barbara, CA) in air modes. All images are presented as height images. For XRD characterization, rice straw powders were recorded by an X’Pert PRO MPD diffractometer (Smartlab(9), Rigaku Corporation) at an accelerating voltage of 45 kV and an emission current of 20 mA with Cu Kα radiation. The diffracted intensity was


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measured in a 2θ range over 10-50° with a step sizes of 0.02° and step times of 1 s. For destruction of cellulose crystallites, rice straw powders were further milled with a PM200 planetary ball mill (Retsch) for 7 h at 650 rpm. Approximately 10 mg of powder was pretreated by 10% NaOH solution and [Emim][OAc], respectively. MD simulation details. MD simulations for cellulose and different solvents were carried out and analyzed in Gromacs 4.6.529. For the ILs the force field developed by Liu et al30 was used and for cellulose bunch we used the Glycam06 force field31. The cellulose was put in the middle of a box surrounded by enough ILs. The initial system was first minimized by the steepest descent method and then was equilibrated for 500ps under NVT ensemble at 373K. After that it was a 100ns dynamics process under NPT ensemble. Long-range electrostatics interactions were calculated of by Particle-mesh Ewald summation32 with a cutoff radius of 1.2nm, which was also the cutoff value for VDW interactions. To mimic a bulk system, periodic boundary conditions were used in all directions. Enzymatic hydrolysis analysis. The destarched cell wall residues (10 mg) were dissolved in 200 µL IL at 90°C. The destarched cell wall residues was pretreated in ILs for different times (5 min, 10 min, 15 min, 20 min, and 30 min), the pretreated rice straw sample was obtained by pelleting in 1 mL 50% acetone + 50% H2O (v/v), and washed by ethyl alcohol to prevent the sample from clumping. After centrifuging the solution at 12000 rpm for 5 min, the pretreated rice straw was freeze-dried to maintain a constant weight for enzymolysis. Approximately 1 mg of pretreated sample was heated in 900 µL deionized water at 100 °C for 1 h. After cooling to room temperature, 100 µL commercial cellulase (Novozymes) solution (1 filter paper activity in 500 mM citrate buffer, pH 4.8) was added to conduct enzymatic hydrolysis in a thermocontrolled shaking incubator at 55 °C for 115 rpm (in a reciprocating shaker). The hydrolytes


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were developed with 2, 4-dinitrosalicyclic acid (DNS), and the absorbance values were recorded at 540 nm. The released levels of reducing sugars were determined by using D-glucose as a standard. NMR spectroscopy analysis. The samples were dissolved in DMSO-d6, and transferred to 5-mm NMR tubes to detect the 1H chemical shifts. The spectra were acquired on a Bruker 600 MHz NMR spectrometer equipped with an inverse gradient 5-mm TXI 1H/13C/15N cryoprobe (proton coils closest to the sample). The chemical shifts were referenced to the central DMSO-d6 solvent peak (δH 2.50 ppm). The NMR data processing and analysis were performed using MestReNova, a product of Mestrelabs Research, SL (Santiago de Compostela, Spain). RESULTS AND DISCUSSION Cellulose microfibrils are swelled and dissolved by [Emim][OAc]. A microfibril is composed of many glucan chains, and the diameter of a microfibril was reported to be approximately 5 nm.26, 33 AFM possesses nanometer resolution and was therefore used to image the wall alterations, especially the major component microfibrils. To reveal the microfibrils pattern of rice cell walls, sections of rice internodes were delignified with sodium chlorite + hydrogen chloride solution and then were observed by AFM under air mode. The microfibrils of rice straw (wild type material) were observed to have a porously aligned network, compared to the dense and neat microfibrils that characterize microbial crystalline cellulose (MCC) (Figure S1). Furthermore, most microfibrils were intertwined into microfibers with diameters of approximately 13.0-15.5 nm in the delignified slices of the normal rice straw. To determine the IL effects on microfibrils, two types of rice straw materials (bc1 and the wild-type with varied cellulose crystallinity statuses were pretreated by [Emim][OAc] at 90 °C


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and then imaged by AFM (Figure 1). A previous study demonstrated that bc1 plants have a reduced cellulose crystallite width and a decreased relative crystallinity index (RCI).34 The bc1 walls were more sensitive to [Emim][OAc] treatment than the wild-type cell walls. Before the treatment, the wild-type and bc1 samples were probed by AFM at room temperature in air-phase mode (Figure 1D-F), showing that most microfibrils in bc1 are isolate and approximately 5.3-8.0 nm in diameter, in contrast to the approximately 13.0-15.5 nm diameters of wild-type microfibrils. After treatment with [Emim][OAc] at 90 °C for 30 min, the microfibrils swelled to approximately 14 nm in bc1 and approximately 21 nm in wild-type (Figure 1G). Therefore, the expansion of microfibrils was striking upon IL treatment. Cellulose microfibrils are main components of plant cell walls, to observe the effect of microfibrils swelling on the dissolution of plant cell wall residues by ILs, rice internodes were selected for IL treatment by confocal imaging. Sclerenchyma cells, tracheids, and parenchyma cells were swelled by [Emim][OAc] treatments at ambient temperature, the thicknesses of the cell walls were greatly changed under different conditions (Figure S2, S3). Cellulose microfibrils are swelled and dissolved by different solvents. The properties of solvents is important factors for dissolving cellulose. Firstly, strong interaction among the cation and anion of the solvent and cellulose should be formed, and the ILs with various hydrogen bonding accept ability (β) and the hydrogen bond acidity (α) have different cellulose dissolving capacities;3,

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Secondly, appropriate size of the solvent is another important factor, only

appropriate size cation or anion could insert into the gap of the glucan chains, then the cellulose microfibers could be swelled effectively, and the cellulose would be dissolved in ILs finally. Brandt et al reported that the IL [C4C1im][MeCO2] with high β value and appropriate size could more effectively swell wood chips than that of [C4C1im][Me2PO4].37 When the cations of ILs


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were different and the anion was [CH3COO]-, the cellulose solubility increased with the increase of α parameter of the ILs.36 Considering the properties of ionic liquids, thus, we selected the common used ionic liquid [Emim][OAc] with appropriate β, α values38 and size as the main solvent in this work. To compare with [Emim][OAc], other different kinds of solvents are also used to pretreat cellulose microfibrils and cell walls. The cross sections of rice internodes were also treated with 10% sodium hydroxide (NaOH) or 85% phosphoric acid (H3PO4) (Figure S4). The walls of sclerenchyma cells, tracheids, and parenchyma cells swelled in response to both solutions within several minutes, however, the cell walls were not dissolved by extending the pretreatment time. Therefore, the expansion of the cell walls is not specific to IL treatment. However, microfibrils not obviously swell in the NaOH and H3PO4 aqueous solution (Figure 2). Moreover, the NaOH solution could strip the hemicellulose from the microfibrils and distort the microfibrils but could not swell microfibrils effectively in short pretreatment time. In addition, glacial acetic acid (CH3COOH) (99.5%) is also used to pretreat cellulose microfibrils (Figure 2), microfibrils swelling degree in glacial acetic acid is less than that in [Emim][OAc], which may indicates that the partial ionization of OAc- from acetic acid induces the cellulose microfibrils swelling, similarly to the OAc- in [Emim][OAc]. In addition, the cross sections of rice internodes and microfibrils were also treated with 1butyl-3-methylimidazolium hexafluorophosphate ([Bmim][PF6]) (Figure S5, Figure 3). Little difference in the cell walls and diameter of microfibrils indicate that [Bmim][PF6] almost has no effect on swelling and dissolving cell walls and microfibrils, which is consistent with that cellulose could not be dissolved in [Bmim][PF6].2


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Simultaneously, to further demonstrate that swelling is the critical process in cellulose dissolution, MCC was treated by [Emim][OAc], 10 % NaOH, CH3COOH and [Bmim][PF6] at 90 °C. Expansion of MCC in those different solvents were observed by the Polarization Microscope (Figure S6). The results indicated that [Emim][OAc] could dissolved MCC completely rapidly but not in 10 % NaOH, CH3COOH and [Bmim][PF6], probably coinciding with that microfibrils only could be effectively swelled in [Emim][OAc]. Thus, microfibrils swelling is necessary for cellulose dissolution. Effect of microfibrils swelling on cellulose crystallinity. To dissect the swelling process in detail, a time-lapse experiment was performed. Microfibrils of delignified wild-type material were imaged by AFM, and wild-type materials were probed by XRD after treatment with [Emim][OAc] for varied times (5 min, 10 min, 15 min, 20 min, and 30 min). The microfibrils diameters were quantified to evaluate the swelling effect of each treatment. The microfibrils diameters were significantly enlarged within 15 min and plateaued after reaching approximately 19 nm (Figure 3); in contrast, no significant alteration was observed after NaOH treatment (Figure 2D). Further, a 100-ns molecular dynamics (MD) simulation showed a cellulosic glucan bunch (8*14, dp = 8) (a simplified microfibril) lost the organized structure in [Emim][OAc] (Figure 3J, 3M), in contrast to almost no change of the cellulosic glucan bunch in [Bmim][PF6] (Figure 3K, 3N). Such results demonstrated again the swelling effect of [Emim][OAc] on cellulose microfibrils. Crystallinity is another characteristic of cellulose microfibrils. The native plant cellulose, which predominantly exists in Iβ conformation, can be evaluated by a specifically sharp peak at 22.5° and a broad peak at 16.7° and 14.9° in the XRDpattern.39 After being treated with ILs, the swelling and dissolution of cellulose microfibrils indicated the collapse of the crystalline


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structure. To investigate the changes in cellulose crystallinity during IL treatment, rice internode powders were probed by XRD spectroscopy after a series of treatments (Figure 4A, S7). The characteristic peaks at 22.5° and 16.7° of crystalline cellulose were almost eliminated, indicating that the microfibrils crystallites were quickly destroyed preceding the full swelling of microfibrils. In contrast, cellulose crystallinity was not greatly reduced by treatment with 10% NaOH (Figure 4B), although partial crystal transfer from native cellulose Iβ to thermally stable cellulose II was observed. In fact, 10% NaOH could fully dissolved the equivalent amount of amorphous cellulose with almost none crystallinity but not crystalline cellulose, demonstrating that the solubility limit had not been reached. Therefore, cellulose crystallite was destroyed effectively by the IL but not by the alkali. Enzymatic hydrolysis after IL pretreatment was performed to evaluate the effects of crystalline destruction and microfibrils swelling. A series of assays showed that the level of released glucose increased after IL treatment (Figure 4C). These results further implied that the improvement in enzymatic hydrolysis mainly resulted from the loss of cellulose crystallinity and microfibrils swelling. Relationship of cellulose microfibrils swelling and IL-HBs. Native cellulose stacking is mediated by the hydrogen bonding between the 3-OH and 5-O and between the 6-OH and 2-OH of glucosyl residues. To elucidate the effects of ILs at the molecular level, NMR spectroscopy was performed to investigate the interactions between IL and cellulose microfibrils. Cellulose oligosaccharides, including cellobiose and cellohexaose, were characterized by NMR in deuterated DMSO (DMSO-d6) with the IL in a gradient of concentrations (Figure 5), as the hydrogen atoms of glucose showed a fully free status in DMSO-d6 (Figure S8). 1H-NMR spectroscopy spectra showed that certain hydrogen atoms from the IL were shifted upfield. Chemical shifts of the acidic H2 were significantly changed, and H4 and H5 were shifted


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upfield. Moreover, clear chemical shift alterations were observed on the hydroxyl hydrogen (OH) rather than the hydrogens of the glycosyl ring (C-H). Therefore, [Emim][OAc] interacts with glucan through the hydrogens of H2, H4 and H5 of the imidazolium cation and promotes glucan dissolution. No significant alterations were found in the chemical shifts of the carbon and hydrogen atoms in the anion OAc-, and the active oxygen atoms of the anion OAc- were very difficult to be probed by NMR. To reveal the roles of the anions, [Emim]Cl or [Emim][OAc] interactions with a glucan (DP = 8) were subjected to MD simulation analysis. In these MD simulations, hydrogen bonds were determined based on cutoffs for the Hydrogen-Donor-Acceptor angle (30°) and the Donor-Acceptor distance (0.35 nm). The molecular simulation results showed that 1) OAc- and Cl- have interaction energies around -1582.5 kJ/mol and -1373.67 kJ/mol, respectively; the interaction energies between Bmim+, PF6- and glucan chain are around -731.4 kJ/mol and -419.4 kJ/mol, compared to an interaction energy around -146.99 kJ/mol for the hydroxide in aqueous solution; and 2) OAc- are mainly located closer than Cl- from the hydroxyl groups of the glucan, and both OAc- and Cl- have more concentrated distributions than those of Bmim+, PF6- and OH-. Therefore, both the interaction energy and distance indicate that [Emim][OAc] is sufficient to form stronger IL-HBs with the hydroxyl groups of glucan chain, and those stronger IL-HBs possess longer interaction life than traditional HBs. That is to say, those stronger IL-HBs induces the ILs have more chances to interact simultaneously with glucan chains. Comparison of diameters of microfibrils swelled by different solvents was summarized (Fig S9), as the molecular or ionic radius increasing, the microfibrils swelling first increased then decreased. According to the results, NaOH aqueous solution almost has no effect on microfibrils swelling in short treated time. Glacial acetic acid could swell the microfibrils to a little degree,


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but could not dissolve it. Although the sizes of cation and anion are all very large, the interaction between PF6- and glucan chain is very weak. The Emim+ and OAc- together could form stronger IL-HBs with glucan chain and disrupt the hydrogen bonds networks of cellulose more easily, therefore, the microfibrils could be swelled effectively. CONCLUSION Swelling is the key procedure in cellulose dissolution by ILs, and cellulose microfibrils swelling was observed in ILs using AFM. Effective swelling demands for ILs with appropriate sizes and strong interactions with cellulose. The strong synergy interactions induce the cations and anions of ILs to interact with glucan chains of cellulose, the hydrogen bonds between the cellulose chains would be disrupted, new IL-HBs among cations, anions and glucan chain would be formed, the cellulose microfibrils were swelled and dissolved. The cellulose crystallite destruction, and ultimately increase of biomass enzymolysis were both owing to effective cellulose microfibrils swelling in ILs.


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FIGURES

Figure 1. Microscopy analyses of cell wall alterations by [Emim][OAc] at 90°C. AFM microscopy of delignified sections treated with the IL [Emim][OAc] after different pretreatment times showing cell-wall swelling. Wild-type sections (A-C) and brittle culm1 (bc1) sections (DF). Color bars in (A-F) represent the height scale of the AFM images. Scale bars = 100 nm; Statistical analyses of microfibrils diameters of wild-type and bc1 (G). The number that follows the ± sign is a standard deviation (SD).


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Figure 2. AFM of rice straw treated by different solvents at room temperature: in 10 % NaOH aqueous solution for 0min (A), 15 min (B) and 30 min (C); in 10 % H3PO4 aqueous solution for 0min (E), 15 min (F) and 30 min (G); in CH3COOH for 0 min (I), 15 min (J) and 30 min (K), scale bars = 100 nm; the changes to microfibrils diameter of rice straw treated by 10% NaOH for different time (D), 10% H3PO4 for different time (H), and in CH3COOH (L). MD simulation of cellulose bunch (8*14, DP = 8), showing the swelling in 10 % NaOH and glacial acetic acid over 100 ns. The conformations of 14*8 cellulose bunch: the front view at 0 ns (M) and the side view at 0 ns (P); 100 ns in 10 % NaOH: view (N), side view (Q); 100 ns in glacial acetic acid: front view (O), side view (R). The number that follows the ± sign is a standard deviation (SD).


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Figure 3. AFM microscopy and MD simulation of microfibrils swelling. AFM imaging of rice straw treated with [Emim][OAc] (A-C) and [Bmim][PF6] (E-G) at 90°C after treatment times. The changes to microfibrils diameter in [Emim][OAc] (D) and in [Bmim][PF6] (H), showing the microfibrils swelling. MD simulation of cellulose bunch (8*14, DP = 8), showing the swelling in [Emim][OAc] (J, M) and [Bmim][PF6] (K, N) over 100 ns. The conformations of 14*8 cellulose bunch: the front view at 0 ns (I) and the side view at 0 ns (L); 100 ns in [Emim][OAc]: front view (J), side view (M); 100 ns in [Bmim][PF6]: front view (K), side view (N). Scale bars = 100 nm. The number that follows the ± sign is a standard deviation (SD).


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Figure 4. The effects of [Emim][OAc] pretreatment of rice straw. (A) XRD spectrogram of the [Emim][OAc] pretreated rice straw powders after treatment times of 0 min (1), 5 min (2), 10 min (3), 15 min (4), 20 min (5) and 30 min (6); (B) The XRD spectrogram of rice straw powders (1) and after milling (2), rice straw powders after pretreatment with 10% NaOH solution (3), and [Emim][OAc]-pretreated rice straw powders (4); (C) enzymolysis analyses of [Emim][OAc]


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pretreated rice straw powders for different times; The amount of released glucose is expressed as the average per mg powders with standard errors.


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Figure 5. 1H chemical shifts of oligosaccharides in [Emim][OAc] and [Emim]Cl, and Radial Distribution Functions (RDFs) of different solvents around cellulose’s hydroxyl hydrogens. Radial Distribution Functions (RDFs) of O2(OAc-), Cl-([Emim]Cl), Ho-Bmi, Ho-F(PF6), and OH1(OH-) around cellulose’s hydroxyl hydrogens: Solvents are 10% aqueous NaOH solution, [Emim][OAc], [Bmim][PF6] and [Emim]Cl.


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Supporting Information. Experimental section of CLSM imaging and microscope observation. Figures from S1-S9. Corresponding Author *Correspondence should be addressed to S. J. Zhang (E-mail: [email protected]) Tel: +86 10 82627080; Fax: +86 10 82544875 Notes The authors declare no competing financial interest. ACKNOWLEDGMENT This research was supported financially by the Program of National Natural Science Foundation of China (NO. 21606240, NO. 21210006, NO. 21336002, NO. 21476234). ABBREVIATIONS ILs, Ionic liquids; HBs, Hydrogen Bonds; IL-HBs, hydrogen bonds of ILs; AFM, atomic force microscopy; XRD, X-ray diffraction; NMR, nuclear magnetic resonance; MD, molecular dynamics; [Bmim]Cl, 1-butyl-3-methylimidazolium chloride; WT, wild-type; bc1, brittle culm1; NaOH, sodium hydroxide, H3PO4, phosphoric acid; [Bmim][PF6], 1-butyl-3-methylimidazolium hexafluorophosphate;

[Emim][OAc],

1-ethyl-3-methyl-

imidazolium

acetate.

MCC,

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SYNOPSIS: Strong hydrogen bond interactions and appropriate size of ILs are crucial in swelling and dissolving cellulose microfibrils. Table of Contents (TOC)/Abstract Graphic


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Nanoscale Observation of Microfibril Swelling and Dissolution in Ionic

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