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Pretreatment of eastern white pine (Pinus strobes L.) for enzymatic hydrolysis and ethanol production by organic electrolyte solutions Xiaofei Tian, Lars Rehmann, Chunbao Charles Xu, and Zhen Fang ACS Sustainable Chem. Eng., Just Accepted Manuscript • DOI: 10.1021/ acssuschemeng.6b00328 • Publication Date (Web): 29 Mar 2016 Downloaded from http://pubs.acs.org on April 5, 2016

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ACS Sustainable Chemistry & Engineering

Pretreatment of eastern white pine (Pinus strobes L.) for enzymatic hydrolysis and ethanol production by organic electrolyte solutions

Xiaofei Tian 2,3,4, Lars Rehmann 2, Charles (Chunbao) Xu 2, Zhen Fang *1,4

1

2

3

Biomass Group, College of Engineering, Nanjing Agricultural University, 40 Dianjiangtai Road, Nanjing, Jiangsu 210031, China.

Department of Chemical and Biochemical Engineering, Western University, London, ON, N6A, 5B9, Canada. School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, 510006, China 4

Biomass Group, Key Laboratory of Tropical Plant Resources and Sustainable Use, Xishuangbanna Tropical Botanical Garden, Chinese Academy of Sciences, Kunming, 650223, China.

*Author for correspondence Zhen Fang ([email protected], [email protected])

Emails: Xiaofei Tian, ([email protected]) Lars Rehmann ([email protected]) Charles (Chunbao) Xu ([email protected])

Revised submission for ACS Sustainable Chemistry & Engineering March 2016

1 ACS Paragon Plus Environment


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Abstract Organic electrolyte solution (OES), composed of room temperature ionic liquids (RTILs) and polar organic solvent, were applied during the pretreatment of eastern white pine (EWP), one of the most recalcitrant woody biomasses. The influence of various crucial parameters that govern the dissolution and further pretreatment process were examined. A gradual reduction of the crystallinity of cellulose I, fragmentation and fibrillation, as well as lignin redistribution occurred with an increase of χ[AMIM]Cl from 0.1 to 0.9; whereas the content of the cellulose, acid insoluble lignin as well as hemicellulose composition did not change. The efficiency of glucose release from EWP through rapid enzymatic hydrolysis (24-h hydrolysis yield) and the final hydrolysis yield (120-h hydrolysis yield) were improved remarkably by up to 460% and 500% after OES pretreatment. No negative effect of OES pretreatment on downstream ethanol fermentation was observed, and the highest ethanol productivity was 11.04 g ethanol / 100 g EWP (when χ[AMIM]Cl=0.9). Keywords: Ionic liquids; Dimethyl Sulfoxide; Permeability; Bioethanol; Fermentability; Softwood


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Introduction Room temperature ionic liquids (RTILs) processes have been receiving significant attention as a promising pretreatment approach for enabling destruction or fractionation of lignocellulosic biomass, as well as producing high yields of fermentable carbohydrates suitable for the production of biofuels.1 However, swelling and agglomeration of lignocellulosic materials may lead to slow or even incomplete dissolution in the process due to the high viscosity of many RTIL.2 In addition, the high costs of the most efficient imidazolium-cation based RTILs have limited the large-scale industrial deployment in pretreatment processes. Some methods aimed at improving the performance efficiency of the RTILs pretreatment, especially by using addition of compatible organic co-solvents within the RTILs, have been recently conceived and developed.3 In this approach, introducing organic electrolyte solutions (OES) demonstrated an improved type of solvent in processing different biomass materials, such as microcrystal cellulose, rice straw, corncob and eucalyptus wood, improving the subsequent enzymatic saccharification efficiency.46

OES was recognized as a binary homogenous solvent composed of RTIL {such as 1-allyl-3-

methylimidazolium chloride ([AMIM]Cl) or 1-ethyl-3-methylimidazolium acetate ([EMIM] [OAc])} and one kind of aprotic organic solution, which featured hydrogen bond acceptor capacities and strong polarity.7 Due to the dilution by the organic solvent portion, OES typically has lower viscosity than neat RTIL but represent very similar Kamlet-Taft solvent parameters relative to neat RTIL.8 Meanwhile, organic solvents with strong permeability (such as DMSO) made OES with a stronger penetration or swelling effect on lignocellulosic biomass. Dissolution could be carried out instantaneously by drastically reducing the dissolving time from hours in RTILs to a few minutes in OES.4,7



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In previous studies, OES showed a similar effectiveness in biomass pretreatment as neat RTILs, such as efficiently decreasing the resistance of the glucosidic bond towards enzymatic attacks by affecting the crystalline cellulosic structure. The crystallinity of cellulose I in the microcrystalline cellulose was reduced by 98.75% after regeneration from the solutions in which [AMIM]Cl was partially replaced by DMSO. Meanwhile, the 72-h enzymatic hydrolysis yield of OES pre-treated samples had a 7.2-time increase with no significant differences found from that of the material pretreated by neat RTIL.4 After OES pretreatment, the enzymatic hydrolysis yield of eucalyptus wood showed a similar value relative to neat RTILs while the dosage of ionic liquid could be decreased by 20%.5 In general, the advantages of OES as a pretreatment tool are the following: (1) Reduction of the dosage of expensive ionic liquids in the processes; (2) promotion of lignin extraction; and (3) lower viscosity compared to neat RTILs, significantly accelerating the dissolving rate and enhancing the solubility of lignocellulose in the OES system, while reducing the resistance in mechanical stirring, as well as enabling fluidity for industrial pipeline transport.6 In OES systems, the employed RTILs can vary but should provide the unique capacities to either selectively dissolve cellulose or lignin. OES possesses the corresponding properties for different purposes in modifying or fractionating biomass, i.e. reduction of cellulosic crystalline structure, or selective de-lignification, or even both of them.2 The compatible organic co-solvents are usually selected from high polarity solvents which also act as hydrogen bonding acceptors.7-8 DMSO is a highly available solvent with a relatively lower toxicity and high productivity among all the RTIL-intermiscibility solvents.9 It is the most commonly used portion in composing OES for biomass pretreatment so far.


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As mentioned above, preliminary results suggested economic benefits of the application of OES in biomass pretreatment. The general feasibility of applying of OES pretreatment on biomass has been established with a variety of raw materials such as pure cellulose, herbaceous plants and hardwood, etc.4-6 Representing a large amount of renewable resources, softwood exhibited unique structural compositions and distribution patterns relative to other kinds of biomass.10 It was recognized as one of the most recalcitrant biomass against not only enzymatic hydrolysis, but also pretreatment.11 This most challenge but indispensable biomass had not been introduced into OES pretreatment before. Hence, it is important to investigate OES-mediated pretreatment of softwoods and its subsequent enzymatic hydrolysis for carbohydrate production. Furthermore, it should be noted that, integrating an OES pretreatment into the entire “from-biomass-tobioethanol” process to evaluate its effects on the downstream fermentability of the hydrolysate for bioethanol production has not yet been reported in any previous study. This study aims to (1) systematically examine key factors of OES pretreatment {[AMIM]Cl and DMSO} with varying molar portion of IL (χ[AMIM]Cl) on EWP, and (2) evaluate the subsequent enzymatic saccharification as well as fermentability of the hydrolysate. Such results will be essential for future development of the novel softwood-to-bio-ethanol process involving OES pretreatment.

Experimental Raw materials and OES preparation EWP (Pinus strobes L.) was collected from a pulp mill in Thunder Bay, ON, Canada. After refined by a micro fine grinder (IKA, MF10 basic, Germany) by passing through a 80-mesh sieve, the material was vacuum-dried for 24 h at 60 ºC before stored in 4 ºC until use. OES, composed of DMSO ( ≥99%, Alfa Aesar, Ward Hill, MA) and [AMIM]Cl (≥99%, water content 5 ACS Paragon Plus Environment


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＜8000ppm, Shanghai Cheng Jie Chemical Co., Ltd., China＝ with 9 different molar fractions (χ[AMIM]Cl=0.1 to χ[AMIM]Cl=0.9), were prepared according to the reference reported by Tian et al.4 [AMIM]Cl was vacuum-dried at 60°C for 48 h before use.

Biomass dissolving and regeneration Each of 0.30 g EWP powders was dissolved into a 25-mL vial containing 6.00 g of pre-heated OES (χ[AMIM]Cl=0.1 to χ[AMIM]Cl=0.9) along with a 5-mm magnetic stirring bar. All of the vials were heated in a glycerol oil bath at 110 ºC for 60 min. Stirring speed was 550 rpm. OES at χ[AMIM]Cl =0 and χ[AMIM]Cl =1 was recognized as neat DMSO and neat [AMIM]Cl, respectively. Afterwards, the mixtures were removed from the bath and cooled down to 25 ºC. Then 15 mL aqueous acetone solution (25 ºC, 50%, w/w) was added into each vials and the suspensions were homogenized by a vortex mixer for 10 s. Precipitates in the mixtures were collected after centrifugation (Sorvall, Thermo Scientific, Waltham, MA) and subsequently washed four times using 15 ºC de-ionized water to remove any OES residuals. The regenerated EWP fraction (recovered EWP) was dehydrated in a freeze dryer (FreeZone 1L, Labconco, Kansas City, MO) for 12 h before sending to further analysis and enzymatic hydrolysis. Triplicate pretreatment runs were conducted at each χ[AMIM]Cl.

Composition analysis and carbohydrate determination Composition analysis of either the original or recovered EWP materials was conducted following the standard NREL protocols with minor modifications.12 Briefly, 0.06 ± 0.005 g of each dried sample was placed in a 25-mL serum bottle (VWR, Canada) containing 0.0948 ± 0.01 g sulphuric acid (72% w/w, Caledon, Canada) and a 5-mm-length magnetic stirring bar. Mixtures were fully mixed at 30 ºC and 300 rpm for an hour on the single-position digital stirring hotplates (Super-Nuova™ SP 131825, Thermo Scientific, Waltham, MA). The mixture was diluted to 4% 6 ACS Paragon Plus Environment

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(w/w) sulphuric acid through the addition of 16.8 ± 0.01 mL (25 ºC) de-ionized water and placed in an autoclave sterilizer at 121 ºC for 60 ± 1min (Eagle Series 2041 Gravity; Amsco, Erie, PA). The acid-insoluble lignin suspended in the hydrolysates was separated after centrifugation and determine gravimetrically. The hydrolysate was retained and the pH was adjusted to 6.0 through the addition of calcium carbonate (98%, Alfa Aesar, Ward Hill, MA). The solid residuals after acid hydrolysis were oven-dried for acid-insoluble lignin and ash determination. Carbohydrates were determined by an Agilent high performance liquid chromatography (HPLC) (1260, Agilent Inc., Santa Clara, CA) equipped with a refractive index detector (RID). The Agilent Hi-Plex H column (7.7×300 mm, 8% cross linked, particle size 8 µm) was maintained at 60 ºC for carbohydrate separation, the mobile phase was 0.005 M sulphuric acid at 0.7 mL min-1. The temperature of the reference and sample cells in RID was set at 55 ºC. Solutions of monomeric sugars at concentrations of 0.1, 0.5, 1, 2, 5 and 10 g L-1 were respectively prepared as standards (Sigma-Aldrich Co. Ltd., St. Louis, MO). The structural composition of the biomass samples was approximated as follows:

XC =

× ×

× 0.9 × 100%

(1)

Where, XC was the cellulose content (%), CG was the glucose concentration in the hydrolysate (g L-1); RG which was the glucose recovery correction factor, representing the correction for losses due to destruction of glucose during each dilute acid hydrolysis (based on glucose control sample). WB (g) was the mass of materials submitted for each composition analysis and V was the total volume of hydrolysate (L).

XH =

× × × × . × ×.

100%


(2)


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Where, XH was the hemicellulose content (%), CX,A,M was the concentration of xylose, arabinose and mannose in the hydrolysate (g L-1); RX,A,M were the respective recovery correction factors.

XAIL = × 100%

(3)

Where XAIL was the acid insoluble lignin content (%, including ash), WR was the oven dried EWP residuals after 4 % (w/w) acid hydrolysis at 121°C for 60 min. Each value was calculated by the means of the triplicated experiments.

FTIR and X-ray diffraction analysis FTIR spectroscopy was conducted to determine the characteristic absorption peaks and chemical bonds in the biomass samples. Each 5 mg of freezing dried wood samples were loaded on a Nicolet 6700 FTIR Spectroscopy with smart iTR TM ATR accessory (Thermo Scientific, Waltham, MA). Wave numbers were set from 4000-550 cm -1 with a scanning speed of 70 cm -1 s-1. X-ray diffraction (XRD) for cellulosic crystalline degree examination was performed on a PANalytical X'Pert PRO MPD system (PANalytical B.V., Almelo, the Netherlands) with Cu Kα radiation of 1.54 Å at 40 kV and 40 mA. All the biomass samples were mounted in the equal amount of 500 mg. Scanning degrees (2ɵ) ranged from 10° to 40°. In XRD patterns, cellulosic crystalline scatterings are superimposed on their amorphous scattering. With regard to the peak height method, crystallinity index (CI) values were calculated as follows:13

CI =

!

(4)

where I002 is the diffraction intensity of peaks corresponding to lattice plane of 002 (22.6° for cellulose I and 21.7° for cellulose II), and Iam is the intensity of the amorphous background, 8 ACS Paragon Plus Environment

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which has a minimum value between lattice planes of 101 and 002 (18.9° for cellulose I from this study and 16.3° for cellulose II from reported value).14

Surface morphology and phase contrast imaging Changes in morphology of EWP after OES treatment were examined by optical microscopy using a DM4000M, Leica microsystem (Wetzlar, Germany). Photos were captured and then processed by ImageJ (v 1.48, National Institute of Health, USA). AFM imaging was performed in tapping mode on a Nanoscope IIIa Multimode scanning probe microscope (Digital Instruments Inc., Santa Barbara, CA). All images (512 x 512 pixels) were measured in air using silicon cantilever Tap-300 (Ted Pella, INC. Redding, CA) with a resonance frequency of 300 kHz and a force constant of 40 N m-1. Two scans were performed on each sample to assure the reproducibility.

Enzymatic hydrolysis Enzymatic hydrolysis was performed using a commercial enzyme cocktail (Cellic CTec II, Novozymes A/S, Bagsvaerd, Denmark). A 30 mg dry biomass was suspended in 3 mL of 50 mM sodium citrate (>99%, BDH Chemicals Ltd., England)-citric acid (1 M, BDH Chemicals Ltd., England) buffer (pH=5.01) in the presents of 0.3 µL of Triton X-100 Surfactant (OmniPur, Billerica, MA). The enzyme loading was 16.6 FPU g-1 dry EWP. The mixture was incubated at 50 ºC in a 20 mL glass vial with screwed cap (VWR, Canada) in a Multitron incubator (Infors HT, Bottmingen, Switzerland) shaken at 200 rpm. Samples were taken at 24 h and 120 h. 100 µL of hydrolysate supernatant was transferred to a 2mL centrifuge tube containing 900 µL of 0.005 M sulphuric acid solution. The dilutions were



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analysed via HPLC after filtrations through cellulose acetate membrane with pore size of 0.2 µm (VWR, Canada). The hydrolysis yield of cellulose to monomeric sugars in EWP after 24 h and 120 h was calculated as follows: &

"#/ = & × 0.9 × 100%

(5)

'

Where, MG (g kg-1) was the mass of glucose per mass initial biomass recovered in hydrolysate determined by HPLC. And MC (g kg-1) was the total amount of cellulose in the EWP as determined via compositional analysis.

Fermentability study Fermentability tests were carried out at the micro-scale with Saccharomyces cerevisiae Meyen ex E.C. Hansen (DSM 1334, Braunschweig, Germany) in 96-well plates (Greiner Cellstar, Kremsmünster, Austria). Enzymatically hydrolysed solution (72-h enzymatic hydrolysis using 16.6 FPU g-1 EWP) was enriched with extra glucose solution (45 g L-1) to achieve initial glucose concentrations of 32.5±0.5 g L-1 in YPM (yeast-peptone medium). S. cerevisiae was firstly incubated in a 100 mL flask containing 10 mL stock solution (20 g L-1 of glucose, 10 g L-1 of peptone and yeast extract (BD Bacto, Franklin Lakes, NJ), at 30 °C for 12 h. During mid-exponential phase, 1 mL of the culture was transferred into 9 mL of fresh stock solutions for additional 6-h incubation. The exponentially growing yeast was separated from the fermentation broth via centrifugation, washed twice and re-suspended in water to remove residual medium and used as an inoculum. Each well was inoculated with 20 µL of yeast


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suspension, 160 µL of hydrolysate, 10 µL peptone (200 g L-1), and 10 µL yeast extract concentrate (200 g L-1) adding to a total of 200 µL. The plate was sealed using a sterilized polypropylene film (Thermo Scientific, Waltham, MA) with micro pores pierced to allow gas exchange. The plate containing the inoculated samples was incubated in a multimode micro-plate reader (Infinite M 200 Pro, Tecan, Männedorf, Switzerland) equipped with a gas control module at 30 °C for 12 h under orbital shacking with amplitude of 2 mm at 57 rpm. Nitrogen (regular grade, Parxail, Canada) was supplied for restricting the concentration of oxygen at 3% (v/v) in the reader’s chamber. The optical density (OD 600) of the fermentation broth was automatically measured at 600 nm every 10 min in each cell. By the end of cultivation, a 150 µL of sample was diluted with 900 µL double distilled water and analyzed for residual sugar and ethanol concentration via HPLC. Hydrolysate fermentations were carried out in triplicate for each material tested. Ethanol conversion (η, %) was calculated according to Eq. 6.

η =(

() *, (*,, ×.-.

× 100%

(6)

Where, C E (g L-1) was the ethanol concentration in the broth, CG,0 (g L-1) and CG,f were the initial and final glucose concentration in the broth. An overall ethanol yield (YE), representing the amount of ethanol produced by per 100 gram of recovered EWP, was calculated as follows: 1

2

*/' ' YE (g/100 g) = / × 0.51 × .% × .% × 100 g .

Analysis of growth kinetics


(7)


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The optical density readings obtained by the plate reader were converted to dry cell weight (DCW, g L-1) based on a calibration curve obtained for the same yeast strain (data not shown). The DCW vs. time date (68 measurements for each sample in triplicate) were analyzed with the Baranyi and Roberts model for population growth, which has been commonly used in many studies of micro-fermentations.15,16 The model used two coupled differential equations:

(8a)

(8b) Where N was the yeast concentration (g L-1), Nmax was the maximum yeast concentration (g L-1), µmax (h-1) was the theoretical maximum specific growth rate and Q was a parameter describing the adaptation of the culture to the environmental conditions. Eqs. 8a and 8b were solved numerically (4th-5th order adaptive Runge Kutta method) and the three regression parameters [µmax, Nmax and Q0=Q (0)] were obtained via least-square regression. All numerical calculations were performed using Matlab (R2015a, Mathworks, Natick, MA). The lag phase was defined as a delayed response of the microbial population to a sudden change in the environment and can therefore be defined as:17

Ln(1 +

λ=

1 ) Q(0)

µmax

(9)

Results and Discussion Dissolving and regeneration of the EWP 12 ACS Paragon Plus Environment

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Biomass was dissolved in the OES system with a varying molar portion of RTIL (χ[AMIM]Cl). During the dissolving process, all of the OES-biomass suspension became dark brown in colour and flocculated once aqueous acetone was added. The biomass recovery during the pretreatment was very high regardless the amount of IL present: 84% ± 2% recovery at χ[AMIM]Cl = 0 and 0.1, and 91% ± 2% at 0.2 ≤ χ[AMIM]Cl ≤1. The weight loss was inferred mostly as the removal of extractives from the EWP by the polar solvent, which was reported as 5.9-7.0%.18

Characterizations of EWP Results of structural carbohydrate analysis revealed that OES pretreatment had no significant effect on the overall composition of EWP, indicating that neither cellulose, hemicellulose, nor lignin were observably removed during the pretreatment, which is in good agreement with the high biomass recoveries (Supplementary material, Table S1). These results are also consistent with those published previously on hardwood treatment by OES ([EMIM] [OAc] and DMSO).5 Possible changes in the molecular structure of the major compounds were evaluated via the Attenuated Total Internal Reflectance - Fourier Transform Infrared Reflectance (ATR- FTIR). Table 1 showed typical chemical bonds appearing in woody biomass and their respective FTIR absorption wavenumbers. The 13 selected wavenumbers showed no significant difference for any of the samples, irrespective of the pretreatment, as seen from the average transmittance (± standard deviation) of all samples.19-21 Combined with the result of the composition study, it can be concluded that no notable depolymerisation, hydrolysis, oxidation or pyrolytic reactions occurred during the EWP pretreatment, likely due to limited water and oxygen contents, as well as the mild processing conditions employed. It further implied that generation of some chemical compounds, which can be typically generated as the by-products in other types of harsh pretreatment and were highly inhibitory to subsequent fermentative conversion, were likely 13 ACS Paragon Plus Environment


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limited.22 However, these inhibiting compounds produced in the OES pretreatment, still need further investigation. In addition to the changes in the chemical composition, pretreatment of biomass may affect its crystalline structure. Generally low crystallinity was desirable as high crystallinity of cellulose may lead to lower enzymatic digestibility. XRD method is a good relative measurement for characterization of cellulosic crystallinity.13 As cellulose content in the pretreated samples was not significantly different (42.8%-44.3%, w/w), loading a same amount of the pretreated sample contained approximately similar cellulose for XRD analysis. Changing of intensity of peak height at specific 2θ angles of 22.6°, 21.7°, 18.9° and 16.3°, typical of XRD angles of lattice planes of 002, 1014and 101 for cellulose I and cellulose II was observed. With the increase of χ[AMIM]Cl in OES, intensities of these cellulose diffraction peaks became weaken (Supplementary material, Figure S1). Table 2 summarized the calculated cellulosic crystallinities of cellulose I and cellulose II in the OES pretreated EWP. Previous studies showed that using pure [AMIM]Cl could lead to a reduction of the CI by 46.5% for the bagasse biomass. 23 Other RTILs such as 1ethyl-3-methylimidazolium chloride ([EMIM]Cl) was also able to reduce the CI of bagasse and eucalyptus materials by 28.6% and 53.3%, respectively.24 The deformation of crystalline cellulose I structure in the regenerated materials was subject to a higher saccharification yield than untreated substrates. In this study, CI of cellulose I decreased by 32.8%, 39.2%, 38.5% and 41.3% after OES pretreatment with χ[AMIM]Cl from 0.7 to 1.0, respectively. It can therefore be confirmed that OES treatment also had the ability to reduce the high crystallinity of cellulose I component in the lignocellulosic biomass, to an extent depending on the content of IL within the OES. Although cellulose II was considered to represent less recalcitrant than cellulose I, this study did not provide evidence of the regular influence on crystalline structure of cellulose II 14 ACS Paragon Plus Environment

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through the OES pretreatment. The modified amorphous forms of cellulose I were anticipated to have positive effects on its hydrolysis kinetics.14, 25 When observed in the micrometer scale, the pretreated EWP samples exhibited a considerable change in morphology from cylindrical fiber fragments into smaller fiber pieces or irregular fragments (Supplementary material, Figure S2). Fibrils were observed on the fiber wall or at the edge of the fragment cuts for χ[AMIM]Cl ≥ 0.5. These changes were likely due to the dissolution of cellulose and hemicellulose in the OES, followed by re-crystallization after the addition of acetone. Hence, the fragmentation and fibrillation of the recovered EWP were demonstrated. The destruction of the EWP fiber appeared to be severer with an increase of χ[AMIM]Cl. To better understand the surface changes of the recovered EWP with respect to χ[AMIM]Cl, measurement with atomic force microscopy (AFM) was performed under ‘light-tapping’ force (Asp/A0=0.8) conditions (Supplementary material, Figure S3). When χ[AMIM]Cl = 0.2, a weak contrast of the phase image indicated a viscoelastically homogeneous material, which likely corresponded to the dissolved cellulose covered on pretreated EWP wood surfaces after regeneration. However, when χ[AMIM]Cl increased to 0.8, the phase image showed a fair contrast between bright grainy structures and darker EWP wood substrate, which likely referred to an amphiphilic contrast through locally varying tip-sample interaction (hydrophobic lignin vs. hydrophilic cellulose). Moreover, a built up of grainy structures of wavy and filamentous shapes on the surface was clearly observed. The features were not visible in detail in the corresponding topographical images of the same spot. The wavy and filamentous structures might be a result of the micrometer morphology of surface lignin or a mixture of lignin, hemicellulose and extractives. When χ[AMIM]Cl =1.0, the disappearance of bright which contrasted in phase occurred. In this case, likely lignin from the original EWP that was not particularly dissolved in [AMIM]Cl 15 ACS Paragon Plus Environment


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was still present in the deep site of the regenerated EWP wood samples rather than on the surface.26 In addition, as shown in Table 3, it was found that both surface morphological roughness (Rmax, Ra, Rq, obtained from height) and surface area difference (height) increased as a function of increasing χ[AMIM]Cl. The appearance of the grainy structures on the regenerated EWP samples correlated well with the phase roughness data, as well as surface area difference (phase) as shown in Table 3. The increase in height roughness with increasing χ[AMIM]Cl was likely due to the increasing ability to dissolve the evenly arranged fibrils on the EWP wood surface. As the linkages between lignin and hemicelluloses were not believed to be broken down by OES, the lignin re-depositing on the EWP wood fragment matrix was possibly caused by swelling, dissolving and precipitation of the cellulose, lignin or hemicelluloses components. Aqueous acetone was known to perform effectively in separating lignin fragments or lignin monomers from dissolved lignin and cellulose solutions.27 In this work, the acid insoluble lignin composition of the recovered EWP was not significantly reduced, nevertheless [AMIM]Cl was reported to have the ability to dissolve EWP powder as a whole by dissolving both lignin and cellulose composition.28 However, there might be different Lignin-OH linkages with which [AMIM]Cl interacted by forming hydrogen bonds in distinct types of lignocellulosic biomasses.29 At the same time, a higher operation temperature could improve the process, which was beyond the scope of this study but would be in our future work. Biomass characterization thus clearly revealed the reduction of crystallinity of cellulose I and the re-deposition of the lignin on the biomass fragment surfaces in EWP after pretreatment. The EWP fragment structure became looser after OES pretreatment. The FTIR characterization of the


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pretreated EWP revealed that the chemical bond structure and the relative content of the main structural components in the regenerated EWP were only slightly affected by the pretreatment, as similarly reported previously.4 All of these indicated that the OES pretreatment preformed in this work was mainly a physicochemical process.

Enzymatic hydrolysis The OES pre-treated and original EWP were hydrolyzed to fermentable sugars via enzymatic saccharification. Enzymatic hydrolysis was monitored after 24 h and 120 h. The hydrolysis yields only slightly increased between 24 h and 120 h (Figure 1). It can also be seen that the hydrolysis yield gradually increased with increasing χ [AMIM] Cl from 0.1 to 0.9. A 67% - 460% and 152% - 500% improvement was achieved over the original EWP, for 24 h (9.5 ± 0.02%) and 120 h (10.5 ± 0.2%), respectively. The highest 24-h hydrolysis yield for the recovered EWP was achieved by OES with χ [AMIM] Cl = 0.8, (yield = 53.0 ± 1.3%). Similar observations were made for the 120-h hydrolysis. Hydrolysis yields increased from 26.4 ± 2.0% to 61.4 ± 2.52 % with the increase of χ [AMIM] Cl from 0.1 to 0.9. The highest yield of 63.0 ± 2.3 % was achieved when χ [AMIM] C l=

0.8. Moreover, there is no significant differences (p

Pretreatment of Eastern White Pine (Pinus strobes ... - ACS Publications

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